Endophytic Microbial Symbionts in Plant Prenatal Care

ABSTRACT

The present disclosure provides compositions comprising novel endophytes capable of promoting germination endophytes that have a symbiotic relationship with plants. The present disclosure further provides methods of improving seed vitality, biotic and abiotic stress resistance, plant health and yield under both stressed and unstressed environmental conditions, comprising inoculating a seed with the novel endophyte strains and cultivating a plant therefrom.

This application is a continuation-in-part of co-pending InternationalApplication No. PCT/CA2013/000091, filed Feb. 5, 2013, which is hereinincorporated in its entirety by reference

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Feb. 4, 2015, is named28761US_sequencelisting.txt, and is 9,382 bytes in size.

FIELD

The present disclosure relates to synthetic preparations comprising aseed and a composition, where the composition comprises fungal andbacterial endophytes of plants that enhance seed vitality and/or planthealth, conferring general improvements in the plant's agriculturaltraits, under normal and stressed conditions.

BACKGROUND

Fungi and bacteria are ubiquitous microorganisms. Endophyte is the termfirst coined by de Bary [1866] defining those microbes that colonizeasymptomatically plant tissues [Stone et al., 2000]. The existence ofendophytes has been known for more than one century [Freeman 1904] andit seems that each individual host, among the 300,000 plant species,inhabits several to hundreds of endophytes [Tan and Zou, 2001].Endophytes are microbial organisms mostly symbiotically ormutualistically associated with living tissues of plant hosts. Many arecapable of conferring plant tolerance to abiotic stressors or can beused by the plant for defense against pathogenic fungi and bacteria[Singh et al. 2011]. Some of these microorganisms have proven useful forvery small subsets of agriculture (e.g., forage grass growth), forestryand horticulture sectors, as well as plant production of medicinallyimportant compounds. However, no commercial endophyte seed coatingproducts are used in the world's largest crops including corn, wheat,rice, and barley, and such endophyte approaches have suffered from highvariability, inconsistent colonization, low performance across multiplecrop cultivars, and the inability to confer benefits to elite cropvarieties under field conditions.

Endophytes largely determine plant cell and whole plant genomeregulation, including the plant's vital cycles: (i) seed pre- andpost-germination events (mycovitalism) [Vujanovic and Vujanovic 2007],(ii) plant nutrient uptake and growth-promoting mechanisms(mycoheterotrophism) [Smith and Read 2008], and (iii) plantenvironmental stress tolerance and induced systemic resistance againstdiseases and pests (mycosymbionticism) [Wallin 1927; Margulis, 1991].They could play a major role in plant biomass production, CO₂sequestration, and/or yield and therefore be significant players inregulating the ecosphere, ensuring plant health and food security. Inaddition, they can be important sentinels (bioindicators) ofenvironmental changes, as alterations in the structure and biomass ofendophytic communities can herald changes not only in pathways ofnutrient (N, P, K), energy transfer in food-webs and biogeochemicalcycles but also in UV-B, heat, drought or salt tolerance influencing theoverall plant ecosystem establishment and stability. Despite theirabundance and likely importance in all terrestrial ecosystems, nearlynothing about the composition of endophytes in seeds or spermosphere,their interactions, or their common response to environmental changes isknown.

While the spermosphere represents a rapidly changing andmicrobiologically dynamic zone of soil surrounding a germinating seed[Nelson, 2004], the rhizosphere is a microbiologically active zone ofthe bulk soil surrounding the plant's roots [Smith and Read 2008]. Therhizosphere supports mycoheterotrophy or a plant-mycorrhiza symbioticrelationship. The spermosphere, on the other hand, promotes mycovitalityor an endophytic fungi relationship with the plant seeds—enhancing seedvigour, energy and uniformity of germination that could be fairlypredicted. Fungal endophytes are distinct from mycorrhizae in that theycan colonize not only roots, but also other plant organs including seeds[Vujanovic et al. 2000; Hubbard et al. 2011]. They belong to themulticellular phyla Ascomycota and Basidiomycota and form colonizationsymbiotic structures different from those produced by unicellular orcenocytic phylum Glomeromycota, known as vesicular-arbuscularmycorrhizal symbiosis [Abdellatif et al. 2009]. Endophytic bacteria havebeen also found in virtually every plant studied, where they colonize anecological niche similar to that of fungi, such as the internal healthytissues. Although most bacterial endophytes appear to originate from therhizosphere or phyllosphere; some may be transmitted through the seed[Ryan et al. 2008].

Seed germination is a vital phenophase to plants' survival andreproduction in either optimal or stressful environmental conditions.Microbial endophytic colonization at the seed state is especiallycritical because of the role of the seed as a generative organ inregeneration and dispersion of flowering plants [Baskin and Baskin 2004]and the role of mycobionts and symbiotically associated bacteria(bactobionts) as potential drivers of seedling recruitment innatural—undisturbed, disturbed and polluted—habitats [Mühlmann andPeintner 2000; Adriaensen et al. 2006; White and Torres 2010]. Thus,developing methods by which seedling emergence can be enhanced andprotected under the limitations of disease pressure, heat or drought isprecious. The use of endophytic symbionts is a promising method by whichseed germination can be enhanced [Vujanovic et al. 2000; Vujanovic andVujanovic 2006; Vujanovic and Vujanovic 2007]. The methods andcompositions described herein overcome these and other limitations ofthe prior art. It was hypothesized that plant stress hardiness can beconferred via a mycobiont-seed relationship known as mycovitality—aphenomenon that had been reserved for Orchidaceae [Vujanovic 2008] andvia bactovitality which refers to a form of bactosymbiosis, usingdifferent endophytic strains with variety of activities.

SUMMARY

The synthetic preparations and compositions described herein can benefitplant hosts, for example, but not limited to, wheat, barley, corn,soybeans, alfalfa, rice, cotton, pulses, canola, vegetables, sugarbeet,sugarcane, trees, shrubs or grasses. The benefit may come frombactovitality, mycovitality and mycoheterotrophy, and enhance toleranceto environmental stresses, as demonstrated herein. Prenatal care inagriculture is more than just seed or germinant vitality, health orvigour. It also determines what to expect before and during thegermination process, seedling establishment, and, later cropproductivity or yield.

Several parameters of symbiotic efficacy (dormancy breakdown,germination, growth and yield) were assessed using efficient endophyticSaskatchewan Microbial Collection and Database (SMCD) strain(s)-crop(s)interaction(s) under in vitro, phytotron, greenhouse and fieldconditions. The synthetic preparations and compositions described hereinhave effects on germination, which can be assessed by measuring percentof germination, energy of germination and hydrothermal time required forgermination, for example.

Also tested was the endophyte's capacity to confer seed vitality. Forboth fungal and bacterial endosymbionts, improved seed vitality canincrease tolerance for abiotic and biotic stresses in plants that haveprogressed beyond the seedling stage to the plant's maturity viamycoheterotrophy. The synthetic preparations and compositions describedherein can improve plant traits such as increased yield, faster seedlingestablishment, faster growth, increased drought tolerance, increasedheat tolerance, increased cold tolerance, increased salt tolerance,increased tolerance to pests and diseases, for example increasedtolerance to Fusarium infection and to Puccinia infection, increasedbiomass, increased root length, increased fresh weight of seedlings,increased plant vigour, nitrogen stress tolerance, enhanced Rhizobiumactivity, enhanced nodulation frequency or early flowering time.

The synthetic preparations and compositions described herein can alsomodulate the expression of genes involved in plant growth, genesassociated with systemic acquired resistance, or genes involved inprotection from oxidative stress. These genes may be involved inphytohormone production, for example gibberellin (GA) biosynthesis orbreakdown, abscisic acid (ABA) biosynthesis or breakdown, NO productionor breakdown, superoxide detoxification, or could be positive ornegative regulators of these pathways. The genes associated withsystemic acquired resistance may be redox-regulated transcriptionfactors, for example those in the MYB family. Non-limiting examples ofsuch genes include ent-kaurenoic (KAO), repression of shoot growth(RSG), NCED, ABA 8′-hydroxylase, GA3-oxidase 2, 14-3-3 or nitric oxide(NO) genes and/or stress resistance superoxide dismutase (SOD),manganese SOD (MnSOD), proline (Pro), Myb1 and Myb2.

In certain embodiments, the present disclosure provides a syntheticpreparation comprising an agricultural plant seed and a compositioncomprising an endophyte capable of promoting germination and anagriculturally-acceptable carrier, wherein an agricultural plant grownfrom the seed has an altered trait as compared to a control agriculturalplant. In certain embodiments, the endophyte capable of promotinggermination is a coleorhiza-activating endophyte and the agriculturalplant seed is a monocot seed. In some embodiments, the composition isdisposed on an exterior surface of the agricultural seed in an amounteffective to colonize the cortical cells of an agricultural plant grownfrom the seed and to produce the altered trait, wherein the alteredtrait is an improved functional trait selected from the group consistingof increased yield, faster seedling establishment, faster growth,increased drought tolerance, increased heat tolerance, increased coldtolerance, increased salt tolerance, increased tolerance to Fusariuminfection, increased tolerance to Puccinia infection, increased biomass,increased root length, increased fresh weight of seedlings, increasedplant vigor, nitrogen stress tolerance, enhanced Rhizobium activity,enhanced nodulation frequency, and early flowering time. In someembodiments, the composition is disposed on an exterior surface of theagricultural seed in an amount effective to colonize at least 1% of thecortical cells of an agricultural plant grown from the seed. In otherembodiments, the composition is disposed on an exterior surface of theagricultural seed in an amount effective to cause a population of seedsinoculated with said composition to have greater germination rate,faster dormancy breakdown, increased energy of germination, increasedseed germination vigor or increased seed vitality than a population ofcontrol agricultural seeds. In some embodiments, the composition isdisposed on an exterior surface of the agricultural seed in an amounteffective to cause a population of seeds inoculated with saidcomposition to reach 50% germination faster than a population of controlagricultural seeds.

In some embodiments, the endophytes are a selected from the groupconsisting of a spore-forming endophyte, a facultative endophyte, afilamentous endophyte, an endophyte capable of living within anotherendophyte, an endophyte capable of forming hyphal coils within theplant, an endophyte capable of forming microvesicles within the plant,an endophyte capable of forming micro-arbuscules within the plant, anendophyte capable of forming hyphal knots within the plant, an endophytecapable of forming Hartig-like nets within the plant, and an endophytecapable of forming symbiosomes within the plant. In some embodiments,the endophyte is in the form of at least one of conidia, chlamydospore,and mycelia.

In other embodiments, the composition is disposed on an exterior surfaceof the agricultural seed in an amount effective to colonize the corticalcells of an agricultural plant grown from the seed and to produce thealtered trait, wherein the altered trait is altered gene expression,wherein the gene is selected from the group consisting of a geneinvolved in plant growth, an acquired resistance gene, and a geneinvolved in protection from oxidative stress. In some embodiments, thegene is involved in phytohormone production. In other embodiments, thegene is a redox-regulated transcription factor. In yet otherembodiments, the gene involved in superoxide detoxification or in NOproduction or breakdown.

In some embodiments, the agricultural plant seed is selected from thegroup consisting of corn, soy, wheat, cotton, rice, canola, barley andpulses. In some embodiments, a population comprising at least 10synthetic preparations is disposed within a packaging material.

Further provided herein is a seed comprising an endophyte or culturedisclosed herein. In one embodiment, the seed is coated with theendophyte. In another embodiment, the seed is cultured or planted nearthe endophyte such that the endophyte is able to colonize the seed. Inone embodiment, the seed planted near the endophyte is up to 4 cm awayfrom the endophyte.

The endophytes used to inoculate the seeds may be selected from thegroup consisting of a spore-forming endophyte, a facultative endophyte,a filamentous endophyte, and an endophyte capable of living withinanother endophyte. In some embodiments, the endophyte is capable offorming certain structures in the plant, where the structures areselected from the group consisting of hyphal coils, Hartig-like nets,microvesicles, micro-arbuscules, hyphal knots, and symbiosomes. In someembodiments, the endophyte is in the form of at least one of conidia,chlamydospore, and mycelia. In other embodiments, the fungus or bacteriais capable of being part of a plant-fungus symbiotic system orplant-bacteria symbiotic system that produces altered levels ofphytohormones or anti-oxidants, as compared to a plant that is not insymbiosis. In other embodiments, the plant-fungus symbiotic system orplant-bacterium symbiotic system has anti-aging and/or anti-senescenceeffects, as compared to a plant or plant organ that is not in symbiosis.In other embodiments, the plant-fungus symbiotic system orplant-bacteria symbiotic system has increased protection againstpathogens, as compared to a plant that is not in symbiosis. In someembodiments,

The present disclosure also provides methods for improving seed vitalityand enhancing plant health and yield under normal and stressedconditions. Accordingly, there is provided a method of improving seedvitality, plant health and/or plant yield comprising inoculating a seedwith an endophyte or culture disclosed herein or a combination ormixture thereof or with a composition disclosed herein. In someembodiments, the seed is cultivated into a first generation plant.

In certain embodiments, provided herein are methods of altering a traitin an agricultural plant seed or an agricultural plant grown from saidseed, where the methods comprise inoculating the seed with a compositioncomprising endophytes capable of promoting germination and anagriculturally-acceptable carrier, where the endophyte replicates withinat least one plant tissue and colonizes the cortical cells of saidplant. In some embodiments, the endophyte colonizes at least 1% of thecortical cells of said agricultural plant.

In some embodiments, the trait altered by using the method is animproved functional trait selected from the group consisting ofincreased yield, faster seedling establishment, faster growth, increaseddrought tolerance, increased heat tolerance, increased cold tolerance,increased salt tolerance, increased tolerance to Fusarium infection,increased tolerance to Puccinia infection, increased biomass, increasedroot length, increased fresh weight of seedlings, increased plant vigor,nitrogen stress tolerance, enhanced Rhizobium activity, enhancednodulation frequency, and early flowering time. In other embodiments,the altered trait is a seed trait selected from the group consisting agreater germination rate, faster dormancy breakdown, increased energy ofgermination, increased seed germination vigor or increased seed vitalitythan a population of control agricultural seeds. In other embodiments,the altered trait is reaching 50% germination faster than a populationof control agricultural seeds. In other embodiments, the altered traitis altered gene expression, where the gene is selected from the groupconsisting of a gene involved in plant growth, an acquired resistancegene, and a gene involved in protection from oxidative stress.

In some embodiments, the endophytes used in the method are a selectedfrom the group consisting of a spore-forming endophyte, a facultativeendophyte, a filamentous endophyte, an endophyte capable of livingwithin another endophyte, an endophyte capable of forming hyphal coilswithin the plant, an endophyte capable of forming microvesicles withinthe plant, an endophyte capable of forming micro-arbuscules within theplant, an endophyte capable of forming hyphal knots within the plant, anendophyte capable of forming Hartig-like nets within the plant, and anendophyte capable of forming symbiosomes within the plant. In someembodiments, the endophyte is in the form of at least one of conidia,chlamydospore, and mycelia.

In some embodiments, the endophyte is a fungus of subphylumPezizomycotina. In some embodiments, the endophyte is a fungus of classLeotiomycetes, Dothideomycetes, Sordariomycetes, or Eurotiomycetes. Insome embodiments, the endophyte is of order Helotiales, Capnodides,Pleosporales, Hypocreales, or Eurotiales. In some embodiments, thecomposition comprises an agriculturally-acceptable carrier and aspore-forming, filamentous bacterial endophyte of phylum Actinobacteria.In some embodiments, the endophyte is a bacteria of orderactinomycetales.

In some embodiments, the present disclosure provides a compositioncomprising a carrier and an endophyte of Paraconyothirium sp. straindeposited as IDAC 081111-03 or comprising a DNA sequence with at least97% identity to SEQ ID NO:5; an endophyte of Pseudeurotium sp. straindeposited as IDAC 081111-02 or comprising a DNA sequence with at least97% identity to SEQ ID NO:4; an endophyte of Penicillium sp. straindeposited as IDAC 081111-01 or comprising a DNA sequence with at least97% identity to SEQ ID NO:3; an endophyte of Cladosporium sp. straindeposited as IDAC 200312-06 or comprising a DNA sequence with at least97% identity to SEQ ID NO:1; an endophyte of Sarocladium sp. straindeposited as IDAC 200312-05 or comprising a DNA sequence with at least97% identity to SEQ ID NO:2; and/or an endophyte of Streptomyces sp.strain deposited as IDAC 081111-06 or comprising a DNA sequence with atleast 97% sequence identity to SEQ ID NO:6. In certain embodiments, theendophyte of Paraconyothirium sp. strain comprises a DNA sequence withat least 98% identity to SEQ ID NO:5; the endophyte of Pseudeurotium sp.strain comprises a DNA sequence with at least 98% identity to SEQ IDNO:4; the endophyte of Penicillium sp. strain comprises a DNA sequencewith at least 98% identity to SEQ ID NO:3; the endophyte of Cladosporiumsp. strain comprises a DNA sequence with at least 98% identity to SEQ IDNO:1; the endophyte of Sarocladium sp. strain comprises a DNA sequencewith at least 98% identity to SEQ ID NO:2; and the endophyte ofStreptomyces sp. strain comprises a DNA sequence with at least 98%sequence identity to SEQ ID NO:6. In certain embodiments, the endophyteof Paraconyothirium sp. strain comprises a DNA sequence with at least99% identity to SEQ ID NO:5; the endophyte of Pseudeurotium sp. straincomprises a DNA sequence with at least 99% identity to SEQ ID NO:4; theendophyte of Penicillium sp. strain comprises a DNA sequence with atleast 99% identity to SEQ ID NO:3; the endophyte of Cladosporium sp.strain comprises a DNA sequence with at least 99% identity to SEQ IDNO:1; the endophyte of Sarocladium sp. strain comprises a DNA sequencewith at least 99% identity to SEQ ID NO:2; and the endophyte ofStreptomyces sp. strain comprises a DNA sequence with at least 99%sequence identity to SEQ ID NO:6. In certain embodiments, the endophyteof Paraconyothirium sp. strain comprises a DNA sequence of SEQ ID NO:5;the endophyte of Pseudeurotium sp. strain comprises a DNA sequence ofSEQ ID NO:4; the endophyte of Penicillium sp. strain comprises a DNAsequence of SEQ ID NO:3; the endophyte of Cladosporium sp. straincomprises a DNA sequence of SEQ ID NO:1; the endophyte of Sarocladiumsp. strain comprises a DNA sequence of SEQ ID NO:2; and the endophyte ofStreptomyces sp. strain comprises a DNA sequence of SEQ ID NO:6.

In another aspect, there is provided a method of improving plant healthand/or plant yield comprising treating plant propagation material or aplant with an endophyte or culture disclosed herein or a combination ormixture thereof or a composition disclosed herein. In some embodiments,the plant propagation material is cultivated into a first generationplant or the plant is allowed to grow.

In an embodiment, the plant propagation material is any plantgenerative/sexual (seed, generative bud or flower) andvegetative/asexual (stem, cutting, root, bulb, rhizome, tuber,vegetative bud, or leaf) part that has the ability to be cultivated intoa new plant.

In an embodiment, the methods enhance landscape development andremediation. Accordingly, in one embodiment, there is provided a methodof reducing soil contamination comprising treating plant propagationmaterial or a plant with an endophyte or culture disclosed herein or acombination or mixture thereof or a composition disclosed herein; andcultivating the plant propagation material into a first generation plantor allowing the plant to grow. In one embodiment, the soil contaminantis hydrocarbons, petroleum or other chemicals, salts, or metals, such aslead, cadmium or radioisotopes.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating embodiments of the disclosure are given by wayof illustration only, since various changes and modifications within thespirit and scope of the disclosure will become apparent to those skilledin the art from this detailed description and respective drawings anddrawing legends.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will now be described in relation to the drawings inwhich:

FIG. 1 shows the phenotypic appearance of the endophytic fungal strainsSMCD 2204, 2004F, 2206, 2208, and 2210 and bacterial strain SMCD 2215;after 10 days of growth on PDA at 21° C.

FIG. 2A shows the inferred neighbour-joining phylogenetic tree of theCladosporium sp. SMCD 2204 based on ITS rDNA. Numbers at nodes indicatebootstrap support values for 1000 replicates; only values that were >70%are given. Bar indicates 0.01 nucleotide substitutions per site(nucleotide position). FIG. 2B shows the inferred neighbour-joiningphylogenetic tree of the Sarocladium sp. SMCD 2204F based on thesequence of the large subunit of the nuclear ribosomal RNA gene (LSU).Numbers at nodes indicate bootstrap support values for 1000 replicates.Only values that were >70% are given. Bar indicates 0.01 nucleotidesubstitutions per site (nucleotide position).

FIG. 3 shows the inferred neighbour-joining phylogenetic tree of thePenicillium sp. SMCD 2206 based on ITS rDNA. Numbers at nodes indicatebootstrap support values for 1000 replicates; only values that were >70%are given. Bar indicates 0.01 nucleotide substitutions per site(nucleotide position).

FIG. 4 shows the inferred neighbour-joining phylogenetic tree of thePseudeurotium sp. SMCD 2208 based on ITS rDNA. Numbers at nodes indicatebootstrap support values for 1000 replicates; only values that were >70%are given. Bar indicates 0.01 nucleotide substitutions per site(nucleotide position).

FIG. 5 shows the inferred neighbour-joining phylogenetic tree of theConiothyrium strain SMCD 2210 based on ITS rDNA. Numbers at nodesindicate bootstrap support values for 1000 replicates; only values thatwere >70% are given. Bar indicates 0.05 nucleotide substitutions persite (nucleotide position).

FIG. 6 shows the inferred neighbour-joining phylogenetic tree of theStreptomyces sp strain SMCD 2215 based on 16S rDNA. Numbers at nodesindicate bootstrap support values for 1000 replicates; only values thatwere >60% are given. Bar indicates 0.05 nucleotide substitutions persite (nucleotide position).

FIG. 7 shows left compartments of split plates (plant with microbialpartner): healthy phenotypic appearance of wheat when the root is grownin contact with the microbial mats; and right-compartments of splitplates (plant without microbial partner): massive formation of roothairs of wheat due to the plant-fungus association made in the leftcompartments of the split plates.

FIGS. 8 (A) and (C) shows SMCD2206 discontinuous colonization of wheatroot (epidermis and cortex) tissue compared to (B) and (D) which showspathogenic Fusarium graminearum's uniform/continual cell colonization ofwheat root including vascular cylinder.

FIG. 9 shows Ireg index—level of deviation (irregularity) in endophyte(SMCDs) cell form.

FIG. 10 shows Idir index—level of direction changes when colonizingliving plant-host cell.

FIG. 11 shows endophytic hyphae in root of wheat germinant (A—SMCD 2204;B—SMCD 2206; C—SMCD 2210; and D—SMCD-2215) visualized with lactofuchsinstaining and fluorescence microscopy. Symbiotic structures/organs: (D)SMCD 2215 bacterial endophyte mostly formed curly intercellularfilaments, whereas endophytic fungi (Figures to the right) produced:SMCD 2204 intracellular coils and arbuscules, SMCD 2206 intracellularvesicules, and SMCD 2110 intracellular knots.

FIG. 12 shows the appearance of symbiotic germinating wheat seedlingsafter 10 days on moist filter paper at 21° C.

FIG. 13 shows leaf length of germinating wheat seedlings after 10 daysat moisture filter paper at 21° C.

FIG. 14 shows an in vitro inoculation method (A). A 5 mm² agar plug, cutfrom the margin of the parent colony, was placed hyphal side down in thecentre of a 60 mm Petri dish containing potato dextrose agar (PDA)media. Next, five surface-sterilized seeds were placed a distanceequivalent to 48 h hyphal growth from the agar plug and germinated inthe dark. The impact of three seed surface sterilization methods on seedgermination (B). Bars labeled with one or two asterisks (*) aresignificantly, or highly significantly, different from the sameendophyte grown under control conditions (p≦0.05 or p≦0.01,respectively; ANOVA, followed by post-hoc LSD test). Error barsrepresent standard error of the mean (SE).

FIG. 15 shows growth rates of free-living endophytes SMCD 2204, 2206,2208, 2210, and 2215 in vitro on potato dextrose agar (PDA) under heatstress (36° C.), drought (8% polyethylene glycol (PEG) 8000) stress andcontrol conditions for five days and simultaneous heat (36° C.) anddrought (8% PEG) for six days. Bars labeled with one or two asterisks(*) are significantly, or highly significantly, different from the sameendophyte grown under control conditions (p≦0.05 or p≦0.01,respectively; ANOVA, followed by post-hoc LSD test). Error barsrepresent standard error of the mean (SE).

FIG. 16 shows percent germination and fresh weight of seedlings frominitial experiments in which seeds were surface sterilized in 5% sodiumhypochlorite for 3 min. Percent germination of wheat seeds in vitroafter three days on potato dextrose agar (PDA) under heat stress (36°C.), drought stress (8% polyethylene glycol (PEG) 8000) and controlconditions (A, B and C) with the y axis normalized to percentgermination obtained under the same conditions by seeds surfacesterilized in 5% sodium hypochlorite for 1 min. Fresh weight ofseedlings in vitro at seven days on PDA under heat stress, droughtstress and control conditions (D, E and F). Bars labeled with one (*) ortwo asterisks (**) are significantly, or highly significantly, differentfrom the no endophyte control (p≦0.05 or p≦0.01, respectively; ANOVA,followed by post-hoc LSD test). Error bars represent the standard errorof the mean (SE).

FIG. 17 shows percent germination over time of wheat seeds co-culturedwith the endophytes most effective at conferring abiotic stresstolerance (SMCD 2206, 2210 and 2215) compared to uncolonized, unstressedseeds (positive control) and uncolonized, stressed seeds (negativecontrol). Energy of germination (EG) is related to the time, in days (xaxis) at which 50% germination (y axis) is reached. The symbols “▪”,“x”, “∘”, “Δ”, and “□” represent the positive control, SMCD 2206 treatedseeds, SMCD 2210 treated seeds, SMCD 2215 treated seeds and the negativecontrol, respectively. Heat and drought treatments correspond to 36° C.and 8% polyethylene glycol (PEG) 8000, respectively. Error barsrepresent the standard error of the mean (SE). Note: The seeds used inEG determination were from the second round of experiments, and hencesterilized in 5% sodium hypochlorite for one minute, rather than three.

FIG. 18 shows the relationship between hydrothermal time (HTT) requiredto achieve 50% germination for heat and drought alone and 5% germinationfor heat and drought combined (x axis) and percent germination attainedafter seven days (y axis). Germination after seven days and HTT werebased on the results of the second round of experiments. The symbols“▪”, “♦” and “▴” represent seeds exposed to heat (36° C.), drought (8%polyethylene glycol (PEG) 8000) or both heat and drought stress,respectively. The R-squared values associated with the trendlines are0.96, 0.80 and 0.18 for seeds exposed to heat, drought or both heat anddrought stress, respectively. Note: The seeds used to determine percentgermination at seven days and HTT were from the second round ofexperiments, and hence treated with 5% sodium hypochlorite for oneminute, rather than three.

FIG. 19 shows seeds treated or inoculated with SMCD strains demonstrateimprovement in all tested seed germination parameters including seedgermination vigour (SGV) efficacy.

FIG. 20 shows the relationship between drought tolerance efficiency(DTE) values in wheat (A) and barley (B) cultivars without (E−) and with(E+) endophytes, based on the average effect of symbiosis using alltested SMCD isolates, on yield exposed to drought stress in greenhouse.

FIG. 21A shows endophytic (E+) inoculants (SMCD 2206, SMCD 2210, andSMCD 2215) improve kernel yield in wheat genotypes compared to control(E−) treatment (yield g/3 pots). FIG. 21B shows endophytic inoculants(SMCD 2206, SMCD 2210, and SMCD 2215) improve kernel yield in two rowbarley (B_(a)) and six row barley (B_(b)) genotypes (kernel yield:3plants/pot).

FIG. 22 shows (A) Barley-six row AC Metcalfe, from left to the right:Drought (E−), Drought and SMCD 2206 (E+), Control (E−), Control and SMCD2206 (E+); (B) Wheat-Unity cultivar, from left to the right: Drought(E−), Drought and SMCD 2215 (E+), Control (E−), Control and SMCD 2215(E+); (C) Wheat-Verona cultivar, from left to the right: Drought (E−),Drought and SMCD 2215 (E+), Control (E−), Control and SMCD 2215 (E+);and (D) Durum wheat-TEAL, from left to the right: Drought (E−), Droughtand SMCD 2210 (E+), Control (E−), Control and SMCD 2210 (E+).

FIG. 23 shows stem dry weight of (A) chickpeas, (B) lentils, and (C)peas in symbiosis with SMCD endophytes (E+) under heat stress phytotronconditions. Bars labeled with one (*) or two asterisks (**) aresignificantly, or highly significantly, different from the no endophytestressed control (p≦0.05 or p≦0.01, respectively; ANOVA, followed bypost-hoc LSD test).

FIG. 24 shows pods dry weight of of (A) chickpeas, (B) lentils, and (C)peas in symbiosis with SMCD endophytes (E+) under heat stress phytotronconditions. Bars labeled with one (*) or two asterisks (**) aresignificantly, or highly significantly, different from the no endophytestressed control (p≦0.05 or p≦0.01, respectively; ANOVA, followed bypost-hoc LSD test).

FIG. 25 shows roots dry weight of (A) chickpeas, (B) lentils, and (C)peas in symbiosis with SMCD endophytes (E+) under heat stress phytotronconditions. Bars labeled with one (*) or two asterisks (**) aresignificantly, or highly significantly, different from the no endophytestressed control (p≦0.05 or p≦0.01, respectively; ANOVA, followed bypost-hoc LSD test).

FIG. 26 shows stem dry weight of (A) chickpeas, (B) peas, and (C)lentils under drought stress in a greenhouse. Bars labeled with one (*)or two asterisks (**) are significantly, or highly significantly,different from the no endophyte (E−) stressed control (p≦0.05 or p≦0.01,respectively; ANOVA, followed by post-hoc LSD test).

FIG. 27 shows dry weights of (A) chickpeas, (B) peas, and (C) lentilspods in association with an endophyte (E+) under drought stress in thegreenhouse. Bars labeled with one (*) or two asterisks (**) aresignificantly different from the no endophyte (E−) stressed control(p≦0.05 or p≦0.01, respectively; ANOVA, followed by post-hoc LSD test).

FIG. 28 shows roots dry weight of (A) chickpeas, (B) peas, and (C)lentils under drought stress in the greenhouse. Bars labeled with one(*) or two asterisks (**) are significantly, or highly significantly,different from no endophyte (E−) stressed control (p≦0.05 or p≦0.01,respectively; ANOVA, followed by post-hoc LSD test).

FIG. 29 shows (A) Chickpea Vanguard flowering plants bearing pods underdrought stress in a greenhouse—left plant is non-symbiotic (E−) andright plant is symbiotic with strain SMCD 2215 (E+); (B) and (C),Chickpea Vanguard plants bearing pods under drought stress in agreenhouse—(B) non-symbiotic and (C) symbiotic with SMCD 2215.

FIG. 30 shows root nodulation of pea varieties under heat stress in aphytotron: Hendel (Above) and Golden (Below) inoculated (left) anduninoculated (right) with SMCD 2215. Note: in all samples naturalinfection with Rhizobium sp. from pea seeds has been observed.

FIG. 31 shows SMCD2206 and SMCD 2215 considerably increase energy ofseed germination (≧50%) in Glamis (lentil) as a function of time underheat and drought in vitro.

FIG. 32 shows SMCD2206 and SMCD 2215 considerably increase energy ofseed germination (≧50%) in Handel (pea) as a function of time under heatand drought in vitro.

FIG. 33 shows endophytic inoculants (SMCD 2206 and SMCD 2210) improveflax yield under drought conditions in a greenhouse. Different lettersabove the bars indicate statistically significant differences betweensamples (p≦0.05, Kruskal-Wallis test).

FIG. 34 shows endophytic inoculants (SMCD 2206, SMCD 2210, and SMCD2215) improve canola yield under drought conditions in a greenhouse.Different letters above the bars indicate statistically significantdifferences between samples (p≦0.05, Kruskal-Wallis test).

FIG. 35 shows the survival of wheat seeds pre-inoculated in-vitro(plates in above row) and wheat seedlings pre-inoculated in greenhouse(pots in below row) with endophytic SMCD 2206-showing healthy plantgrowth, and with pathogenic Fusarium avenaceum and Fusariumgraminearum—showing disease symptoms and death of plants.

FIG. 36 shows Fusarium inoculants produced on wheat kernels.

FIG. 37 shows that post-emergence damping-off has been prevented by SMCD2206 endophyte in wheat in the greenhouse.

FIG. 38 shows wheat biomass (aerial a-d and root e-f) improved in thepresence of SMCD 2206 endophyte compared to untreated plants. (a)control plant (E−), (b) inoculated plant (E+), (c) control floweringplant, (d) inoculated flowering plant, (e) control plant (E−, left)compared to SMCD 2206 inoculated plant (E+, right), and (f) fluorescentmicroscopy of SMCD 2206 wheat root-colonization (E+).

FIG. 39 shows aerial plant biomass/plant (left) and underground (root)biomass/plant (right) in control (E−) and SMCD inoculated wheat plants(E+) against F. graminearum and F. avenaceum. Vertical error bars ondata points represent the standard error of the mean.

FIG. 40 shows root length in control plant (CDC Teal) without SMCDendophyte compared to inoculated wheat plant with SMCD strains. Bars ondata points represent the standard error of the mean.

FIG. 41 shows dry weight of kernels/plant (TEAL cultivar) in wheat usingthe double pre-inoculation approach: a) SMCD endophyte+Fusariumavenaceum (F.av), and b) SMCD endophyte+Fusarium graminearum (F.gr).Vertical error bars on data points represent the standard error of themean.

FIG. 42 shows comparison of TEAL spike sizes in wheat in the presence ofpathogen (negative control) and without presence of pathogen (positivecontrol). Left Figure—from left to right: i) plant+F.gr, ii) plant+F.av,and (iii) plant; Right Figure—from left to right: i) plant; ii)plant+endophyte; iii) plant+endophyte+F. av; and iv)plant+endophyte+F.gr.

FIG. 43 shows the effect of SMCD 2215 on Handel (pea) on 10% PEG after 7days at 21 degrees C. in darkness. (A) shows the control seeds, and (B)shows the SMCD 2215-treated seeds.

FIG. 44 shows (A) SOD and (B) MnSOD relative gene expressions in pea(Handel) exposed to PEG with and without endophytes.

FIG. 45 shows Proline relative gene expression in pea (Handel) exposedto PEG with and without endophytes.

FIG. 46 shows germination of wheat seeds in vitro after three days onpotato dextrose agar (PDA). Cold stratification was imposed by keepingseeds at 4° C. cold-room for 48 hours. For endophyte-indirect andendophyte-direct treatments, using SMCD 2206, seeds were germinated atapproximately 4 cm distance and in direct contact respectively. A)Percentage of germination in comparison with energy of germination (50%germination). B) Efficacy of germination of wheat seeds subjected tocold and biological stratification. Efficacy was calculated bysubtracting the germination percentage of control from treated seeds.

FIG. 47 shows differential expression patterns of gibberellin (TaGA3ox2and 14-3-3) and ABA (TaNCED2 and TaABA8′OH1) genes in coleorhiza ofgerminating wheat seeds for three days under cold and biologicalstratification. Gene expression was calculated as 2⁻ ^(CT) .

FIG. 48 shows the ratio of expression levels (2⁻ ^(CT) ) of gibberellin(TaGA3ox2 and 14-3-3) and ABA (TaNCED2 and TaABA8′OH1) genes incoleorhiza of germinating wheat seeds for three days under cold andbiological stratification.

FIG. 49 shows relative expression patterns of hormonal RSG and KAOregulator genes and MYB 1 and MYB 2 resistance genes in coleorhiza ofgerminating wheat seeds for three days under cold and biologicalstratification. Gene expression was calculated as 2⁻Δ^(CT).

FIG. 50 shows emerging radicle from wheat geminating seed (A) Invertedfluorescence (B) and fluorescence imaging of DAF-2DA fluorescence uponreaction with NO in radicle cells (C) of AC Avonlea germinant at 5 minafter treatment [Nakatsubo et al. 1998] with the fungal SMCD 2206exudate. No fluorescence reaction observed in control radicle cells.Bar=25 mm; Bar=50 mm.

FIG. 51 shows DAF-2T fluorescence intensity values at 5 min aftertreatment of wheat radicle from AC Avonlea germinants with the SMCD 2206fungal exudate, fungal exudate together with the NO scavenger cPTIO, andsterile water. Radicle segments were incubated for 30 min in 2 ml ofdetection buffer (10 mM Tris-Hcl, pH 7.4, 10 mM KCl) containing 15 μMDAF-2DA (Sigma-Aldrich) with or without 1 mM2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(cPTIO) as an NO scavenger. Average fluorescence values are reported asa ratio of the fluorescence intensity at 5 min to the fluorescenceintensity at time 0. Different letters indicate statisticallysignificant differences between samples (p≦0.05, Kruskal-Wallis test).

FIG. 52 shows the change (in days) in the initial flowering time ofcanola crops treated with the microbial compositions described. Datashown is from n=4 independent replicate plots±one standard deviation.1=Abiotic formulation control, 2=SMCD 2204, 3=SMCD 2204F, 4=SMCD 2206,5=SMCD 2208, 6=SMCD 2210, 7=SMCD 2215.

FIG. 53 shows the damage score due to pests of canola crops treated withthe microbial compositions described. Data shown is from n=4 independentreplicate plots±one standard deviation. 1=Abiotic formulation control,2=SMCD 2204, 3=SMCD 2204F, 4=SMCD 2206, 5=SMCD 2208, 6=SMCD 2210, 7=SMCD2215.

FIG. 54 shows the Fusarium Head Blight (FHB) incidence percentage forthree spring wheat (Lillian, Unity, and Utmost) and one durum wheat(Strongfield) varieties. Subplots (a), (b), (e), and (f) refer to theVanguard, Saskatchewan, Canada test site, while subplots (c), (d), (g),and (h) refer to the Stewart Valley, Saskatchewan, Canada test site.Data shown is from n=4 independent replicate plots±one standarddeviation. 1=Abiotic formulation control, 2=SMCD 2204, 3=SMCD 2204F,4=SMCD 2206, 5=SMCD 2208, 6=SMCD 2210, 7=SMCD 2215.

FIG. 55 shows the leaf spot disease rating for three spring wheat(Lillian, Unity, and Utmost) and one durum wheat (Strongfield)varieties. A rating of 1 is most healthy and a rating of 10 is mostdiseased. Subplots (a), (b), (e), and (f) refers to the leaf spotdisease rating on the lower leaf, while subplots (c), (d), (g), and (h)refer to the leaf spot disease rating on the flag leaf. Data shown isfrom n=4 independent replicate plots±one standard deviation. 1=Abioticformulation control, 2=SMCD 2204, 3=SMCD 2204F, 4=SMCD 2206, 5=SMCD2208, 6=SMCD 2210, 7=SMCD 2215.

FIG. 56 shows aggregated yield data for durum wheat, spring wheat,barley, canola, and pulses (chickpea, pea, and lentil). Each dot refersto a single plot. The “+” refers to the group mean. The data arepresented as percentage gain over the abiotic formulation control foreach combination of crop, location and condition (irrigated or dryland).(a) refers to SMCD 2215, (b) refers to SMCD 2210, (c) refers to SMCD2204, (d) refers to SMCD 2206, (e) refers to SMCD 2208, and (f) refersto SMCD 2204F. Data shown are from n=4 independent replicate plots forall Canadian sites and n=6 plots for Brookings, S. Dak. sites. While nofields were experimentally inoculated with a pathogen, the notation“Fus−” indicates that there was no visible occurrence of Fusarium HeadBlight (Fusarium graminearum) in that specific field and “Fus+”indicates that there was clear evidence of Fusarium Head Blight(Fusarium graminearum) occurrence. N− indicates that there was nonitrogen applied to the field and N+indicates that nitrogen was appliedat agriculturally relevant rates.

FIG. 57 shows aggregated yield data for durum wheat, spring wheat,barley, canola, and pulses (chickpea, pea, and lentil). 95% confidenceintervals for the respective formulation and crop are shown. The dataare presented as percentage gain over the abiotic formulation controlfor each combination of crop, field trial and condition (irrigated ordryland). (a) refers to SMCD 2215, (b) refers to SMCD 2210, (c) refersto SMCD 2204, (d) refers to SMCD 2206, (e) refers to SMCD 2208, and (f)refers to SMCD 2204F. Data shown is from n=4 independent replicate plotsfor all Canadian sites and n=6 plots for Brookings, S. Dak. As above,the notation “Fus-” indicates that there was no visible occurrence ofFusarium Head Blight (Fusarium graminearum) in that specific field and“Fus+” indicates that there was clear evidence of Fusarium Head Blight(Fusarium graminearum) occurrence. N− indicates that there was nonitrogen fertilizer applied to the field and N+ indicates that nitrogenfertilizer was applied at agriculturally relevant rates.

FIG. 58 shows the aggregated ear weight data from the corn field trialin Brookings, S. Dak. 95% confidence intervals for the respectiveformulation is shown. The data is presented as percentage gain over theabiotic formulation control. “2206” refers SMCD 2206, “2204” refers toSMCD 2204, and “2215” refers to SMCD 2215.

FIG. 59 shows data from greenhouse trials of tomato inoculated with thedescribed microbial compositions: (a) shoot length, (b) shoot weight,(c) total plant biomass, (d) root length, (e) root weight, and (f)tomato fruit weight under drought conditions. Data shown is from n=3independent replicate plants±one standard deviation.

FIG. 60 shows data from greenhouse trials of alfalfa treated with thedescribed microbial compositions: (a) shoot length, (b) shoot weight,(c) total plant biomass, (d) root length, and (e) root weight underdrought conditions. Data shown is from n=3 independent replicateplants±one standard deviation.

FIG. 61 shows data from greenhouse trials comparing normal (dark gray)and drought (light gray) water conditions for: (a) corn, (b) sweet corn,(c) organic corn, (d) swiss chard, (e) radish, and (f) cabbage. Datashown is total biomass from n=3 independent replicate plants±onestandard deviation.

FIG. 62 shows gibberellin production data from wheat (CDC Avonlea) inseedling studies. “Control” refers to the formulation control whereinSMCD 2206 was not added, “A-Direct” refers to direct application of SMCD2206 to the seedling, and “A-Indirect” refers to the indirectapplication of SMCD 2206 to the seedling through a small-moleculepermeable membrane. See Table 12 for abbreviation of molecules. Datashown is average concentration in ng per gram dry tissue weight from n=3independent replicates.

FIG. 63 shows ABA metabolite production data from wheat (CDC Avonlea) inseedling studies. “Control” refers to the formulation control whereinSMCD 2206 was not added, “A-Direct” refers to direct application of SMCD2206 to the seedling, and “A-Indirect” refers to the indirectapplication of SMCD 2206 to the seedling through a small-moleculepermeable membrane. See Table 13 for abbreviation of the names of themolecules. Data shown is average concentration in ng per gram dry tissueweight from n=3 independent replicates.

FIG. 64 shows cytokinin production data from wheat (CDC Avonlea) inseedling studies. “Control” refers to the formulation control whereinSMCD 2206 was not added, “A-Direct” refers to direct application of SMCD2206 to the seedling, and “A-Indirect” refers to the indirectapplication of SMCD 2206 to the seedling through a small-moleculepermeable membrane. See Table 14 for abbreviation of molecules. Datashown is average concentration in ng per gram dry tissue weight from n=3independent replicates.

FIG. 65 shows auxin production data from wheat (CDC Avonlea) in seedlingstudies. “Control” refers to the formulation control wherein SMCD 2206was not added, “A-Direct” refers to direct application of SMCD 2206 tothe seedling, and “A-Indirect” refers to the indirect application ofSMCD 2206 to the seedling through a small-molecule permeable membrane.Auxins were represented by the biologically active indole acetic acidIAA and its conjugate with aspartic acid IAA-Asp. Data shown is averageconcentration in ng per gram dry tissue weight from n=3 independentreplicates.

FIG. 66 shows symbiosomes in root of wheat germinant visualized withlactofuchsin staining and fluorescence microscopy. A type I symbiosome,which is composed of an intercellular microvesiculoid compartment formedbetween two plant cell membranes (arrows), a perivesiculoid membrane(large triangle) and a partially fragmented outer vesiculoid membrane(small triangle), is shown in A. A type II symbiosome, which is composedof an intracellular vesiculoid compartment (arrows), a perivesiculoidmembrane (large triangle) and a partially fragmented outer vesiculoidmembrane (small triangle), is shown in B. Also shown in B is avesiculophore (filled arrow). The symbiosomes shown in A and B are bothof vesicle form. Shown in C are symbiosomes of knot form (type I—lowerarrow; type II— upper arrow).

DEFINITIONS

The term “mycovitality” as used herein refers to the plant-fungussymbiosis that exists between the seeds and the fungi, which helpsmaintain the seeds' capacity to live and develop, and leads togermination. In some embodiments, mycovitality may be characterized by achange in levels of certain phytohormones within the plant-fungussymbiotic system. In some embodiments, this change may be associatedwith a change in the levels of abscisic acid (ABA), gibberellins (GA),auxins (IAA), and/or cytokinins. In other embodiments, mycovitality maybe characterized by a change in expression of the genes involved ingibberellin (GA) biosynthesis or breakdown or in abscisic acid (ABA)biosynthesis or breakdown, or in positive or negative regulation ofthese pathways within the plant-fungus symbiotic system. In certainembodiments, the levels of expression of the gibberellin (GA)biosynthetic genes, such as GA3-oxidase 2, RSG, KAO, and 14-3-3 genesmay be increased. In other embodiments, the levels of expression of thegenes that are regulated by GAs, such as ent-kaurenoic (KAO) andrepression of shoot growth (RSG), are increased. In other embodiments,the levels of expression of the GA degradation genes or negativeregulators of the GA biosynthesis pathway, for example 14-3-3 genes, aredecreased. In still other embodiments, mycovitality may be characterizedby decreased levels of expression of the genes involved in the ABAbiosynthesis pathway, for example the NCED gene, within the plant-fungussymbiotic system. In other embodiments, the expression of the genesinvolved in the ABA catabolic pathway, for example the 8′-hydroxylasegene, are increased. In some embodiments, mycovitality may becharacterized by altered levels of nitric oxide (NO) within theplant-fungus symbiotic system, for example as a result of a change inthe expression of certain genes involved in NO production or breakdown.In yet other embodiments, mycovitality may be characterized byprotection of the plant-fungus symbiotic system from oxidative stress.In some embodiments, mycovitality is characterized by increased levelsof expression of the genes involved in superoxide detoxification withinthe plant-fungus symbiotic system. In some embodiments, the genesassociated with superoxide detoxification encode superoxide dismutase(SOD) or manganese SOD (MnSOD), and in other cases the levels of theamino acid proline (Pro) are elevated. In some embodiments, mycovitalityis characterized by an increase in the levels of activity of the genesassociated with systemic acquired disease resistance, such asredox-regulated transcription factors, for example those in the MYBfamily. In some embodiments, the genes in the MYB family are Myb1 andMyb2.

The term “bactovitality” as used herein refers to the plant-bacteriumsymbiosis that exists between the seeds and the bacteria, which helpsmaintain the seeds' capacity to live and develop, and leads togermination. In some embodiments, bactovitality may be characterized bya change in levels of certain phytohormones within the plant-bacteriumsymbiotic system. In some embodiments, this change may be associatedwith a change in the levels of abscisic acid (ABA), gibberellins (GA),auxins (IAA), and/or cytokinins. In other embodiments, bactovitality maybe characterized by a change in expression of the genes involved ingibberellin (GA) biosynthesis or breakdown or in abscisic acid (ABA)biosynthesis or breakdown, or in positive or negative regulation ofthese pathways within the plant-bacteria symbiotic system. In certainembodiments, the levels of expression of the gibberellin (GA)biosynthetic genes, such as GA3-oxidase 2, RSG, KAO, and 14-3-3 genesmay be increased. In other embodiments, the levels of expression of thegenes that are regulated by GAs, such as ent-kaurenoic (KAO) andrepression of shoot growth (RSG), are increased. In other embodiments,the levels of expression of the GA degradation genes or negativeregulators of the GA biosynthesis pathway, for example 14-3-3 genes, aredecreased. In still other embodiments, bactovitality may becharacterized by decreased levels of expression of the genes involved inthe ABA biosynthesis pathway, for example the NCED gene, within theplant-bacterium symbiotic system. In other embodiments, the expressionof the genes involved in the ABA catabolic pathway, for example the8′-hydroxylase gene, are increased. In some embodiments, bactovitalitymay be characterized by altered levels of nitric oxide (NO) within theplant-bacterium symbiotic system, for example as a result of a change inthe expression of certain genes involved in NO production or breakdown.In yet other embodiments, bactovitality may be characterized byprotection of the plant-bacterium symbiotic system from oxidativestress. In some embodiments, bactovitality is characterized by increasedlevels of expression of the genes involved in superoxide detoxificationwithin the plant-bacterium symbiotic system. In some embodiments, thegenes associated with superoxide detoxification encode superoxidedismutase (SOD) or manganese SOD (MnSOD), and in other cases the levelsof the amino acid proline (Pro) are elevated. In some embodiments,bactovitality is characterized by an increase in the levels of activityof the genes associated with systemic acquired disease resistance, suchas redox-regulated transcription factors, for example those in the MYBfamily. In some embodiments, the genes in the MYB family are Myb1 andMyb2.

“Cold stratification” as used herein refers to the process ofpretreating seeds to simulate the natural winter conditions duringwhich, amongst many physiological changes, the seed coat is softened upby frost and weathering action, leading to dormancy breakdown.“Biological stratification” as used herein refers to the process oftreating seeds with biological components to release seed dormancy andthereby promoting germination. In some embodiments, the biologicalcomponents may be endophytes. Therefore, as compared to coldstratification, in which an abiotic stimulation is used, biologicalstratification uses a biotic stimulation. As for cold stratification,biological stratification may increase the rate of germination in seeds.In both cases, the progress of stratification and dormancy breakdown maybe associated with an increase in levels of GA and a decrease in levelsof ABA. In certain embodiments, the levels of expression of gibberellin(GA) biosynthetic genes, such as GA3-oxidase 2 and 14-3-3 genes, areincreased. In other embodiments, the levels of expression of the genesthat are regulated by GAs, such as ent-kaurenoic (KAO) and repression ofshoot growth (RSG), are increased. In other embodiments, the levels ofexpression of GA degradation genes or negative regulators of the GAbiosynthesis pathway, for example 14-3-3 genes, are decreased. In stillother embodiments, the levels of expression of the genes involved in theABA biosynthesis pathway, for example the NCED gene, are decreased. Inother embodiments, the expression of genes involved in the ABA catabolicpathway, for example the 8′-hydroxylase gene, is increased.

“Anti-aging” or “anti-senescence” as used herein refers to a processwithin a seed or plant that protects the seed or plant from aging andsenescence or that results in delayed aging or senescence of the seed orplant. In some embodiments, the anti-aging or anti-senescence effects ofendophytes are characterized by increased levels of nitric oxide (NO)within the plant-fungus or plant-bacterium symbiotic system, for exampleas a result of a change in the expression of certain genes involved inNO production or breakdown. In certain embodiments, the anti-aging oranti-senescence effects of endophytes may be characterized by a changein levels of certain phytohormones within the plant-fungus orplant-bacterium symbiotic system. In some embodiments, this change maybe associated with decreased by levels of abscisic acid (ABA), increasedlevels of gibberellins (GA) or increased levels of auxins. In someembodiments, mycovitality may be characterized by a change in expressionof the genes involved in gibberellin (GA) biosynthesis or breakdown orin abscisic acid (ABA) biosynthesis or breakdown, or in positive ornegative regulation of these pathways within the plant-fungus orplant-bacterium symbiotic system. In certain embodiments, the levels ofexpression of the gibberellin (GA) biosynthetic genes, such asGA3-oxidase 2, RSG, KAO, and 14-3-3 genes may be increased. In otherembodiments, the levels of expression of the genes that are regulated byGAs, such as ent-kaurenoic (KAO) and repression of shoot growth (RSG),are increased. In other embodiments, the levels of expression of the GAdegradation genes or negative regulators of the GA biosynthesis pathway,for example 14-3-3 genes, are decreased. In still other embodiments, theanti-aging or anti-senescence effects of endophytes may be characterizedby decreased levels of expression of the genes involved in the ABAbiosynthesis pathway, for example the NCED gene, within the plant-fungusor plant-bacterium symbiotic system. In other embodiments, theexpression of the genes involved in the ABA catabolic pathway, forexample the 8′-hydroxylase gene, are increased.

As used herein, “symbiosome” or “symbiotic organs” refers to the newcompartment that is formed within the plant cell when bacteria or fungicolonize the plant. In type I symbiosomes, the new structure is anintercellular microvesiculoid compartment formed between two plant cellmembranes. A “microvesiculoid” compartment is a structure that has theform of a microvesicle. In type II symbiosomes, the new compartment islocalized intracellularly and can be described as an intracellularstructure in the form of a vesicle, or “intracellular vesiculoidcompartment.” Both types of symbiosomes are further characterized by thepresence of a “perivesiculoid membrane,” which is the plasma membranethat surrounds the vesicles, and a partially fragmented “outervesiculoid membrane,” which is an outer membrane in the form of avesicle. In this context, a symbiosome is not limited to the structurethat is formed during nitrogen fixation.

“Mycoheterotrophy” as used herein refers to a symbiotic relationshipbetween a plant and a fungus that allows the plant to obtain water,minerals, and carbohydrates more efficiently. In this context, the plantmay be any plant, even a fully photosynthetic plant, that may derive abenefit via its association with the fungus.

As used herein, the term “microarbuscule” refers to intracellular,multiarbuscular, microsized (˜10 um), bush-like haustorial structures.

The term “vitality,” as used herein means the capacity to live anddevelop.

The term “hydrothermal time” refers to parameters of water, temperatureand time by which seed germination can be described under variousenvironmental conditions. The parameters enable germination strategiesto be compared in different environments and to assess the effects ofendophytes on germination relative to other variables.

In some embodiments, the endophyte is chosen from the group consistingof a spore-forming endophyte, a facultative endophyte, a filamentousendophyte, and an endophyte capable of living within another endophyte.In some embodiments, the endophyte is capable of forming certainstructures in the plant, where the structures are selected from thegroup consisting of hyphal coils, Hartig-like nets, microvesicles,micro-arbuscules, hyphal knots, and symbiosomes. In some embodiments,the endophyte is in the form of at least one of conidia, chlamydospore,and mycelia. In other embodiments, the fungus or bacteria is capable ofbeing part of a plant-fungus symbiotic system or plant-bacteriasymbiotic system that produces altered levels of phytohormones oranti-oxidants, as compared to a plant that is not in symbiosis. In otherembodiments, the plant-fungus symbiotic system or plant-bacteriumsymbiotic system has anti-aging and/or anti-senescence effects, ascompared to a plant or plant organ that is not in symbiosis. In otherembodiments, the plant-fungus symbiotic system or plant-bacteriasymbiotic system has increased protection against pathogens, as comparedto a plant that is not in symbiosis.

A “spore” or a population of “spores” refers to bacterial or fungalstructures that are more resilient to environmental influences such asheat and bacteriocidal agents and fungicides than vegetative forms ofthe same bacteria or fungi. Spores are typically capable of germinationand out-growth giving rise to vegetative forms of the species. Bacteriaand fungi that are “capable of forming spores” or “spore-formingendophytes” are those bacteria and fungi containing the genes and othernecessary abilities to produce spores under suitable environmentalconditions.

The term “filamentous fungi” as used herein are fungi that form hyphae,and includes taxa that have both filamentous and yeast-like stages intheir life cycle.

The term “facultative endophytes” as used herein are endophytes capableof surviving in the soil, on the plant surface, inside a plant and/or onartificial nutrients. Facultative endophytes may also have the capacityto survive inside a variety of different plant species.

The term “endophyte capable of living within another endophyte” as usedherein refers to an endophytic bacterium or fungus that can live withinanother endophyte. Such endophytic bacteria may also be able to liveautonomously in the soil, on the plant surface, inside a plant and/or onartificial nutrients.

The term “endophyte capable of promoting germination” as used hereinrefers to endophytes that have the capacity to colonize a seed or partof a seed and alter the seed's physiology such that the seed or apopulation of seeds shows a faster dormancy breakdown, greatergermination rate, earlier germination, increased energy of germination,greater rate of germination, greater uniformity of germination,including greater uniformity of rate of germination and greateruniformity of timing of germination, and/or increased vigor and energyof germination. In some embodiments, the endophyte capable of promotinggermination is an endophyte that is capable of activating the coleorhizaof a monocot seed, and can be called a “coleorhiza-activatingendophyte”.

The term “agricultural plant” means a plant that is typically used inagriculture. The agricultural plant may be a monocot or dicot plant, andmay be planted for the production of an agricultural product, forexample grain, food, fiber, etc. The plant may be a cereal plant. Theterm “plant” as used herein refers to a member of the Plantae Kingdomand includes all stages of the plant life cycle, including withoutlimitation, seeds, and includes all plant parts. The plant can beselected from, but not limited to, the following list:

Food crops: Cereals including Maize/corn (Zea mays), Sorghum (Sorghumspp.), Millet (Panicum miliaceum, P. sumatrense), Rice (Oryza sativaindica, Oryza sativa japonica), Wheat (Triticum sativa), Barley (Hordeumvulgare), Rye (Secale cereale), Triticale (Triticum X Secale), Oats(Avena fatua);

leafy vegetables (brassicaceous plants such as cabbages, broccoli, bokChoy, rocket; salad greens such as spinach, cress, lettuce);

fruiting and flowering vegetables (e.g. avocado, sweet corn, artichokes,curcubits e.g. squash, cucumbers, melons, courgettes, pumpkins;solononaceous vegetables/fruits e.g. tomatoes, eggplant, capsicums);

podded vegetables (groundnuts, peas, beans, lentils, chickpea, okra);

bulbed and stem vegetables (asparagus, celery, Allium crops e.g garlic,onions, leeks);

roots and tuberous vegetables (carrots, beet, bamboo shoots, cassava,yams, ginger, Jerusalem artichoke, parsnips, radishes, potatoes, sweetpotatoes, taro, turnip, wasabi);

sugar crops including sugar beet (Beta vulgaris), sugar cane (Saccharumofficinarum);

crops grown for the production of non-alcoholic beverages and stimulants(coffee, black, herbal and green teas, cocoa, tobacco);

fruit crops such as true berry fruits (e.g. kiwifruit, grape, currants,gooseberry, guava, feijoa, pomegranate), citrus fruits (e.g. oranges,lemons, limes, grapefruit), epigynous fruits (e.g. bananas, cranberries,blueberries), aggregate fruit (blackberry, raspberry, boysenberry),multiple fruits (e.g. pineapple, fig), stone fruit crops (e.g. apricot,peach, cherry, plum), pip-fruit (e.g. apples, pears) and others such asstrawberries, sunflower seeds;

culinary and medicinal herbs e.g. rosemary, basil, bay laurel,coriander, mint, dill, Hypericum, foxglove, alovera, rosehips);

crop plants producing spices e.g. black pepper, cumin cinnamon, nutmeg,ginger, cloves, saffron, cardamom, mace, paprika, masalas, star anise;

crops grown for the production of nuts and oils e.g. almonds andwalnuts,

Brazil nut, cashew nuts, coconuts, chestnut, macadamia nut, pistachionuts; peanuts, pecan nuts, soybean, cotton, olives, sunflower, sesame,lupin species and brassicaeous crops (e.g. canola/oilseed rape); and,

crops grown for production of beers, wines and other alcoholic beveragese.g grapes, hops;

edible fungi e.g. white mushrooms, Shiitake and oyster mushrooms;

Plants Used in Pastoral Agriculture:

legumes: Trifolium species, Medicago species, and Lotus species; Whiteclover (T. repens); Red clover (T. pratense); Caucasian clover (T.ambigum); subterranean clover (T. subterraneum); Alfalfa/Lucerne(Medicago sativum); annual medics; barrel medic; black medic; Sainfoin(Onobrychis viciifolia); Birdsfoot trefoil (Lotus corniculatus); GreaterBirdsfoot trefoil (Lotus pedunculatus);

Forage and Amenity grasses: Temperate grasses such as Lolium species;Festuca species; Agrostis spp., Perennial ryegrass (Lolium perenne);hybrid ryegrass (Lolium hybridum); annual ryegrass (Lolium multiflorum),tall fescue (Festuca arundinacea); meadow fescue (Festuca pratensis);red fescue (Festuca rubra); Festuca ovina; Festuloliums (Lolium XFestuca crosses); Cocksfoot (Dactylis glomerata); Kentucky bluegrass Poapratensis; Poa palustris; Poa nemoralis; Poa trivialis; Poa compresa;Bromus species; Phalaris (Phleum species); Arrhenatherum elatius;Agropyron species; Avena strigosa; Setaria italic;

Tropical grasses such as: Phalaris species; Brachiaria species;Eragrostis species; Panicum species; Bahai grass (Paspalum notatum);Brachypodium species; and,

Grasses used for biofuel production such as Switchgrass (Panicumvirgatum) and Miscanthus species;

Fiber Crops:

hemp, jute, coconut, sisal, flax (Linum spp.), New Zealand flax(Phormium spp.); plantation and natural forest species harvested forpaper and engineered wood fiber products such as coniferous andbroadleafed forest species;

Tree and Shrub Species Used in Plantation Forestry and Bio Fuel Crops:

Pine (Pinus species); Fir (Pseudotsuga species); Spruce (Picea species);Cypress (Cupressus species); Wattle (Acacia species); Alder (Alnusspecies); Oak species (Quercus species); Redwood (Sequoiadendronspecies); willow (Salix species); birch (Betula species); Cedar (Cedurusspecies); Ash (Fraxinus species); Larch (Larix species); Eucalyptusspecies; Bamboo (Bambuseae species) and Poplars (Populus species).

Plants Grown for Conversion to Energy, Biofuels or Industrial Productsby Extractive, Biological, Physical or Biochemical Treatment:Oil-producing plants such as oil palm, jatropha, linseed;

Latex-producing plants such as the Para Rubber tree, Hevea brasiliensisand the Panama Rubber Tree Castilla elastica;

plants used as direct or indirect feedstocks for the production ofbiofuels i.e. after chemical, physical (e.g. thermal or catalytic) orbiochemical (e.g. enzymatic pre-treatment) or biological (e.g. microbialfermentation) transformation during the production of biofuels,industrial solvents or chemical products e.g. ethanol or butanol,propane diols, or other fuel or industrial material including sugarcrops (e.g. beet, sugar cane), starch-producing crops (e.g. C3 and C4cereal crops and tuberous crops), cellulosic crops such as forest trees(e.g. Pines, Eucalypts) and Graminaceous and Poaceous plants such asbamboo, switch grass, miscanthus;

crops used in energy, biofuel or industrial chemical production bygasification and/or microbial or catalytic conversion of the gas tobiofuels or other industrial raw materials such as solvents or plastics,with or without the production of biochar (e.g. biomass crops such asconiferous, eucalypt, tropical or broadleaf forest trees, graminaceousand poaceous crops such as bamboo, switch grass, miscanthus, sugar cane,or hemp or softwoods such as poplars, willows; and,

biomass crops used in the production of biochar;

Crops Producing Natural Products Useful for the Pharmaceutical,Agricultural Nutraceutical and Cosmeceutical Industries:

crops producing pharmaceutical precursors or compounds or nutraceuticaland cosmeceutical compounds and materials for example, star anise(shikimic acid), Japanese knotweed (resveratrol), kiwifruit (solublefiber, proteolytic enzymes);

Floricultural, Ornamental and Amenity Plants Grown for their Aestheticor Environmental Properties: Flowers such as roses, tulips,chrysanthemums;

Ornamental shrubs such as Buxus, Hebe, Rosa, Rhododendron, Hedera;

Amenity plants such as Platanus, Choisya, Escallonia, Euphorbia, Carex;

Mosses such as sphagnum moss; and

Plants Grown for Bioremediation:

Helianthus, Brassica, Salix, Populus, and Eucalyptus.

A “host plant” includes any plant, particularly an agricultural plant,which an endophytic microbe such as an endophyte capable of promotinggerminations can colonize. As used herein, a microbe is said to“colonize” a plant or seed when it can be stably detected within theplant or seed over a period time, such as one or more days, weeks,months or years. In other words, a colonizing microbe is not transientlyassociated with the plant or seed.

As used herein, an “agricultural seed” is a seed used to grow a planttypically used in agriculture (an “agricultural plant”). The seed may beof a monocot or dicot plant, and may be planted for the production of anagricultural product, for example grain, food, fiber, etc. The seed maybe of a cereal plant. As used herein, an agricultural seed is a seedthat is prepared for planting, for example, in farms for growing.

As used herein, a “control agricultural plant” or “control seed” is anagricultural plant or seed of the same species, strain, or cultivar towhich a treatment, formulation, composition or endophyte preparation asdescribed herein is not administered/contacted. A control agriculturalplant or control seed, therefore, is identical to the treated plant orseed with the exception of the presence of the endophyte and can serveas a control for detecting the effects of the endophyte that isconferred to the plant.

A “population” of plants or seeds, as used herein, can refer to aplurality of plants or seeds that were subjected to the same inoculationmethods described herein, or a plurality of plants or seeds that areprogeny of a plant or group of seeds that were subjected to theinoculation methods. In addition, a population of plants can be a groupof plants that are grown from coated seeds. The plants or seeds within apopulation will typically be of the same species, and will alsotypically share a common genetic derivation.

The term “endophyte” as used herein refers to a fungal or bacterialorganism that can live symbiotically in a plant and is also referred toherein as “endosymbiont”. A fungal endophyte may be in the form of aspore, hypha, or mycelia. A bacterial endophyte may be a cell or groupof cells. The term “endophyte” as used herein includes progeny of thestrains recited herein.

In some cases, the present invention contemplates the use of microbesthat are “compatible” with agricultural chemicals, for example, afungicide, an anti-bacterial compound, or any other agent widely used inagricultural which has the effect of killing or otherwise interferingwith optimal growth of microbes. As used herein, a microbe is“compatible” with an agricultural chemical when the microbe is modified,such as by genetic modification, e.g., contains a transgene that confersresistance to an herbicide, or is adapted to grow in, or otherwisesurvive, the concentration of the agricultural chemical used inagriculture. For example, a microbe disposed on the surface of a seed iscompatible with the fungicide metalaxyl if it is able to survive theconcentrations that are applied on the seed surface.

As used herein, a “colony-forming unit” (“CFU”) is used as a measure ofviable microorganisms in a sample. A CFU is an individual viable cellcapable of forming on a solid medium a visible colony whose individualcells are derived by cell division from one parental cell.

In some embodiments, the invention uses microbes that are heterologousto a seed or plant in making synthetic combinations or agriculturalformulations. A microbe is considered heterologous to the seed or plantif the seed or seedling that is unmodified (e.g., a seed or seedlingthat is not treated with a population of endophytes capable of promotinggermination described herein) does not contain detectable levels of themicrobe. For example, the invention contemplates the syntheticcombinations of seeds or seedlings of agricultural plants and anendophytic microbe population (e.g., an endophyte capable of promotinggermination), in which the microbe population is “heterologouslydisposed” on the exterior surface of or within a tissue of theagricultural seed or seedling in an amount effective to colonize theplant. A microbe is considered “heterologously disposed” on the surfaceor within a plant (or tissue) when the microbe is applied or disposed onthe plant in a number that is not found on that plant before applicationof the microbe. For example, population of endophytes capable ofpromoting germination that is disposed on an exterior surface or withinthe seed can be an endophyte that may be associated with the matureplant, but is not found on the surface of or within the seed. As such, amicrobe is deemed heterologously disposed when applied on the plant thateither does not naturally have the microbe on its surface or within theparticular tissue to which the microbe is disposed, or does notnaturally have the microbe on its surface or within the particulartissue in the number that is being applied. The term “exogenous” can beused interchangeably with “heterologous.”

The phrase “inoculating a seed” as used herein refers to applying,infecting, co-planting, spraying, immersing, dusting, dipping or coatingthe seed with the endophyte. Techniques for inoculating the seed areknown in the art, for example, as disclosed by Hynes and Boyetchko(2006, Soil Biology & Biochemistry 38: 845-84). In an embodiment,inoculation comprises foliar application or soil application of theendophyte or combination thereof with any solid or liquid carrier at anygrowing stage of the plant.

The term “enhancing seed vitality” as used herein refers to plantprenatal care improving the ability of the seed to germinate and producea plant under normal and/or stressed conditions and includes, withoutlimitation, any one or more of the following: breaking dormancy,providing seed stratification, increasing seed germination, modulatinggene expression, decreasing time to reach energy of germination,protecting against biotic stresses, protecting against abiotic stresses,reducing hydrothermal time required for germination, increasing seedgermination vigour, increasing seed germination efficacy, increasinguniformity of seed germination, ameliorating drought/heat toleranceefficacy, increasing the weight of seedlings, and increasing the yieldof seedlings. Drought/Heat Tolerance Efficiency (DTE/THE) is the termopposed (antonym) to susceptibility.

Energy of germination is defined as 50% of germination, relative to thenumber of seeds tested. The seed germination vigour shows the differencebetween total percentage of germinating treated seeds and germinatinguntreated seeds. The hydrothermal time postulates that an individualseed begins to germinate when the sum of both temperatures and waterpotential are sufficiently accumulated over a period of time allowinggermination. Germination efficacy is defined as the percentage oftreated seeds germinating after a set time period after planting,relative to the number of seeds tested in an untreated control.Biological stratification is defined as releasing seed dormancy by asymbiont in promoting germination. Uniformity of seed germinationrepresents the maximum percentage of seed germination within a minimaltime of incubation.

The terms “decreased”, “fewer”, “slower” and “increased” “faster”“enhanced” “greater” as used herein refers to a decrease or increase ina characteristic of the endophyte treated seed or resulting plantcompared to an untreated seed or resulting plant. For example, adecrease in a characteristic may be at least 1%, at least 2%, at least3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 75%, at least 100%, or at least 200% or more lowerthan the untreated control and an increase may be at least 1%, at least2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 75%, at least 100%, or at least 200%or more higher than the untreated control.

The term “increased yield” refers to increased seed weight, seed size,seed number per plant, seed number per unit area (i.e. seeds, or weightof seeds, per acre), bushels per acre, tons per acre, kilo per hectare,increased grain yield, increased dry weight of grain, increased seedweight, increased dry weight of wheat spikes and increased biomass.“Biomass” means the total mass or weight (fresh or dry), at a giventime, of a plant tissue, plant tissues, an entire plant, or populationof plants. Biomass is usually given as weight per unit area. Increasedbiomass includes without limitation increased pod biomass, stem biomass,and root biomass.

In certain embodiments, the plant is cultivated under abiotic or bioticstressed conditions. The term “abiotic stress” as used herein refers toa non-living stress that typically affects seed vitality and planthealth and includes, without limitation, heat, drought, nitrogen, cold,salt and osmotic stress. In one embodiment, the abiotic stress is heatstress. In another embodiment, the abiotic stress is drought stress,osmotic stress or salt stress. The term “biotic stress” as used hereinrefers to a living stress that typically affects seed vitality and planthealth, and includes without limitation, insect infestation, nematodeinfestation, bacterial infection, fungal infection, oomycete infection,protozoal infection, viral infection, and herbivore grazing, or anycombination thereof. In one embodiment, the biotic stress is a Fusariuminfection.

As used herein an “agriculturally compatible carrier” refers to anymaterial, other than water, which can be added to a seed or a seedlingwithout causing or having an adverse effect on the seed (e.g., reducingseed germination) or the plant that grows from the seed, or the like.

The term “plant propagation material” as used herein refers to any plantgenerative/sexual and vegetative/asexual part that has the ability to becultivated into a new plant. In an embodiment, the plant propagationmaterial is generative seed, generative bud or flower, and vegetativestem, cutting, root, bulb, rhizome, tuber, vegetative bud, or leafparts.

In some cases, the present invention contemplates the use of microbes(e.g., endophytes) that are “compatible” with agricultural chemicals,for example, a fungicide, an anti-bacterial compound, or any other agentwidely used in agriculture that has the effect of killing or otherwiseinterfering with optimal growth of microbes. As used herein, a microbesuch as a seed bacterial endophyte is “compatible” with an agriculturalchemical when the microbe is modified, such as by genetic modification,e.g., contains a transgene that confers resistance to an herbicide, oris adapted to grow in, or otherwise survive, the concentration of theagricultural chemical used in agriculture. For example, a microbedisposed on the surface of a seed is compatible with the fungicidemetalaxyl if it is able to survive the concentrations that are appliedon the seed surface.

The term “phytoremediation” as used herein refers to the use of plantsfor removal, reduction or neutralization of substances, wastes orhazardous material from a site so as to prevent or minimize any adverseeffects on the environment. The term “phytoreclamation” as used hereinrefers to the use of plants for reconverting disturbed land to itsformer or other productive uses.

DETAILED DESCRIPTION

The present invention identifies a small, unique family of endophytesthat can be placed into synthetic combination with a variety of planthosts and work synergistically with the plant hosts to exhibit asurprising number of altered and improved biological processes.

Plants across the Angiosperms have many features in common that emanatefrom having evolved from a common ancestor. This is true for the manysystems that control growth and development and also tolerance toabiotic and biotic stresses. Plants have co-evolved with endosymbiontsand in consequence these latter organisms, fungi and bacteria, canpossess features that enable them to interact with plants. It is wellaccepted that microrganisms can be classified on the basis of theirtaxonomy or cladistics groupings, as well as based on key morphological,functional, and ecological roles. Here, by screening hundreds ofsynthetic associations between endophytes and plants, we discovered afamily of endophytes based on their ability to interact with a varietyof plant species to create agricultural value. These endophytes possesssystems that enable them to physically and chemically interact with abroad range of agricultural plants bred by man, including monocots anddicots, endorsing the conclusion that when living together with theplant they interact intimately with the conserved genetic andphysiological properties of plant species. Our classification based onstudies involving a large range of agricultural plants distinguishes thechosen endophytes from those endophytes that interact with only someclasses of plant species. Endophytes classified in this way can includefungi and bacteria and the classification highlights that the fungi andbacteria have informational systems in common. The informational systemsprogramming the plant-endophyte interactions are complex and comprisesignaling systems, multiple networks and pathways that underpin growthof many types of plant cells and organs as well as endophyte cells. Theyare thus best described by the outcomes of the plant-endophyteinteractions.

The endophyte class described herein provides the unique ability toconfer mycovitalism to a large number of diverse plant hosts, as well asto confer stress tolerance and increased yield. Specifically, thisendophyte class is able to, when coated onto the outside of a seed orplaced in its proximity, increase expression of key genes related toseed germination, vigor, and stress tolerance. The endophytes are thenable to penetrate the cortical layer of the seed and plant in order toenter the plant's internal tissues and replicate within at least onetissue in the host and establish symbiotic organs comprisingmicrostructures that allow intimate communication between the endophyteand the plant's intra- and/or intercellular spaces. These endophytesfurther act in symbiosis with the host to improve stress tolerance ofthe seedling and adult plant and to increase yield.

Germination of mature, dry seeds is a process that is conserved acrossangiosperms, being regulated by water, temperature, the hormonesgibberellic acid, abscisic acid and ethylene, amongst other vitalmolecules, and involves changes to cell walls, breakdown of foodreserves and their conversion to new molecules and structures thatdefine root and shoot growth. The group of endophytes revealed here isreadily characterized by its ability to stimulate seed germination ormake germination more uniform when any of its members are present as asynthetic preparation that physically interacts with a seed from monocotor dicot plants. In other embodiments, the group of endophytes isrecognized as capable of altering plant flowering time and/or increasingtolerance to biotic and abiotic stresses and many other traits. All ofthese features support the conclusion that members of this group ofendophytes can be physically complexed with monocot and dicot seeds toachieve multiple agricultural benefits due to their particularinformational systems that interact with those conserved in plants.

This family of endophytes represents a surprising discovery in theirability to engage in synthetic associations with plants, leading to anumber of altered physiological processes across the lifespan of theplant-endophyte composite association. Notably, the syntheticassociations between this small family of endophytes and both monocotand dicot plants are characterized by the activation of multiple plantgenes and hormones during seed germination, seedling development, andresponses to environmental and biotic stresses.

Novel Compositions and Seeds

Accordingly, the present disclosure provides a composition comprising atleast one endophyte capable of promoting germination or comprising acombination or mixture thereof, and an agriculturally-acceptablecarrier. In some embodiments, the at least one endophyte capable ofpromoting germination are coleorhiza-activing endophytes. In someembodiments, a synthetic preparation is made using the composition andan agricultural plant seed. In some cases, plants are inoculated with atleast one endophyte that is heterologous to the inoculated agriculturalplant seed or the agricultural plant grown from the agricultural seed.In some embodiments, the at least one endophyte capable of promotinggermination are disposed on the surface or within a tissue of theagricultural seed or seedling. In some embodiments, a plant grown from aseed inoculated with this composition has an improved functional traitas compared to a control plant. In some embodiments, the improvedfunctional trait is resistance to biotic or abiotic stress. In someembodiments, the improved functional trait is selected from the groupconsisting of increased yield, faster seedling establishment, fastergrowth, increased photosynthetic rate, increased carbon dioxideassimilation rate, increased drought tolerance, increased heattolerance, increased cold tolerance, increased salt tolerance, increasedtolerance to pests and diseases, increased biomass, increased rootand/or shoot length or weight, increased fresh weight of seedlings,increased seed or fruit number, increased plant vigour, nitrogen stresstolerance, enhanced Rhizobium activity, enhanced nodulation frequency,early flowering time, or any combination thereof. In some embodiments,the increased tolerance to disease is increased tolerance to Fusariuminfection, increased tolerance to Septoria infection, and/or increasedtolerance to Puccinia infection. In some embodiments, yield is measuredon a population of plants grown in the field and is calculated viacombine harvesting or measuring ear weight. For all altered traits, thechange can be at least 1%, for example at least 2%, at least 3%, atleast 4%, at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 75%, at least 100%, ormore, when compared with a control agricultural seed or plant. In someembodiments, the improved trait is heritable by progeny of theagricultural plant grown from the seed.

In some embodiments, the agricultural seed is a seed of a monocot plant.In some embodiments, the agricultural seed is a seed of a cereal plant.In some embodiments, the agricultural seed is a seed of a corn, wheat,barley, rice, sorghum, millet, oats, rye or triticale. In someembodiments, the agricultural seed is a seed of a dicot plant. In someembodiments, the agricultural seed is a seed of cotton, canola, soybeanor a pulse.

In some embodiments, a synthetic preparation is made comprising a canolaseed and a composition comprising at least one endophyte capable ofpromoting germination and an agriculturally-acceptable carrier, and acanola plant grown from the seed flowers earlier as compared to acontrol canola plant. In some embodiments, a synthetic preparation ismade comprising a tomato, alfalfa, corn, swiss chard, radish, or cabbageseed and a composition comprising at least one endophyte capable ofpromoting germination and an agriculturally-acceptable carrier, and atomato, alfalfa, corn, swiss chard, radish, or cabbage plant grown underdrought conditions from the seed has higher biomass as compared to acontrol plant grown under drought conditions.

In some embodiments, the composition is disposed on an exterior surfaceof the agricultural seed in an amount effective to colonize at least0.1%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%,at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70% or at least 80% of cortical cells of a plantgrown from the seed.

In some embodiments, the composition comprises a carrier and at leastone endophyte chosen from the group consisting of a spore-formingendophyte, a facultative endophyte, a filamentous endophyte, and anendophyte capable of living within another endophyte. In someembodiments, the at least one endophyte is capable of forming certainstructures in the plant, where the structures are selected from thegroup consisting of hyphal coils, Hartig-like nets, microvesicles,micro-arbuscules, hyphal knots, and symbiosomes. In some embodiments,the at least one endophyte is in the form of at least one of conidia,chlamydospore, and mycelia. In other embodiments, the fungus or bacteriais capable of being part of a plant-fungus symbiotic system orplant-bacteria symbiotic system that produces altered levels ofphytohormones or anti-oxidants, as compared to a plant that is not insymbiosis. In other embodiments, the plant-fungus symbiotic system orplant-bacterium symbiotic system has anti-aging and/or anti-senescenceeffects, as compared to a plant or plant organ that is not in symbiosis.In other embodiments, the plant-fungus symbiotic system orplant-bacteria symbiotic system has increased protection againstpathogens, as compared to a plant that is not in symbiosis.

In some embodiments, the at least one endophyte is a fungus of subphylumPezizomycotina. In some embodiments, the at least one endophyte is afungus of class

Leotiomycetes, Dothideomycetes, Sordariomycetes, or Eurotiomycetes. Insome embodiments, the at least one endophyte is of order Helotiales,Capnodides, Pleosporales, Hypocreales, or Eurotiales. In someembodiments, the at least one endophyte is selected from one of thefollowing families: Acarosporaceae, Adelococcaceae, Agyriaceae,Aigialaceae, Ajellomycetaceae, Amniculicolaceae, Amorphothecaceae,Amphisphaeriaceae, Amplistromataceae, Anamylopsoraceae, Annulatascaceae,Anteagloniaceae, Antennulariellaceae, Aphanopsidaceae, Apiosporaceae,Apiosporaceae, Arachnomycetaceae, Arctomiaceae, Armatellaceae,Arthoniaceae, Arthopyreniaceae, Arthrodermataceae, Arthrorhaphidaceae,Ascobolaceae, Ascocorticiaceae, Ascodesmidaceae, Ascodichaenaceae,Ascosphaeraceae, Asterinaceae, Aulographaceae, Australiascaceae,Baeomycetaceae, Bambusicolaceae, Batistiaceae, Bertiaceae,Biatorellaceae, Biatriosporaceae, Bionectriaceae, Boliniaceae,Brigantiaeaceae, Bulgariaceae, Byssolomataceae, Caliciaceae,Caloscyphaceae, Calosphaeriaceae, Calycidiaceae, Candelariaceae,Capnodiaceae, Carbomycetaceae, Carbonicolaceae, Catabotrydaceae,Catillariaceae, Celotheliaceae, Cephalothecaceae, Ceratocystidaceae,Ceratomycetaceae, Ceratostomataceae, Chadefaudiellaceae, Chaetomiaceae,Chaetosphaerellaceae, Chaetosphaeriaceae, Chaetosphaeriaceae,Chaetothyriaceae, Chorioactidaceae, Chrysotrichaceae, Cladoniaceae,Cladosporiaceae, Clavicipitaceae, Clypeosphaeriaceae, Coccocarpiaceae,Coccodiniaceae, Coccoideaceae, Coccotremataceae, Coenogoniaceae,Collemataceae, Coniocessiaceae, Coniochaetaceae, Coniocybaceae,Coniothyriaceae, Cordycipitaceae, Coronophoraceae, Coryneliaceae,Corynesporascaceae, Crocyniaceae, Cryphonectriaceae, Cryptomycetaceae,Cucurbitariaceae, Cudoniaceae, Cyphellophoraceae, Cyttariaceae,Dactylosporaceae, Davidiellaceae, Delitschiaceae, Dermateaceae,Diademaceae, Diaporthaceae, Diatrypaceae, Didymellaceae,Didymosphaeriaceae, Discinaceae, Dissoconiaceae, Dothideaceae,Dothidotthiaceae, Dothioraceae, Ectolechiaceae, Elaphomycetaceae,Elixiaceae, Elsinoaceae, Eremascaceae, Eremithallaceae, Erysiphaceae,Euceratomycetaceae, Extremaceae, Fissurinaceae, Fuscideaceae,Geoglossaceae, Glaziellaceae, Gloeoheppiaceae, Glomerellaceae,Glomerellaceae, Gnomoniaceae, Gomphillaceae, Gondwanamycetaceae,Graphidaceae, Graphostromataceae, Gyalectaceae, Gymnoascaceae,Gypsoplacaceae, Haematommataceae, Halojulellaceae, Halosphaeriaceae,Halotthiaceae, Harknessiaceae, Helminthosphaeriaceae, Helotiaceae,Helvellaceae, Hemiphacidiaceae, Heppiaceae, Herpomycetaceae,Herpotrichiellaceae, Hyaloscyphaceae, Hymeneliaceae, Hypocreaceae,Hyponectriaceae, Hypsostromataceae, Icmadophilaceae, Jobellisiaceae,Juncigenaceae, Karstenellaceae, Kathistaceae, Koerberiaceae,Koralionastetaceae, Laboulbeniaceae, Lachnaceae, Lasiosphaeriaceae,Lecanoraceae, Lecideaceae, Lentitheciaceae, Leotiaceae, Leprocaulaceae,Leptosphaeriaceae, Letrouitiaceae, Lichinaceae, Lindgomycetaceae,Lobariaceae, Lophiostomataceae, Lophiotremataceae, Loramycetaceae,Lulworthiaceae, Lyrommataceae, Magnaporthaceae, Malmideaceae,Massariaceae, Massarinaceae, Megalariaceae, Megalosporaceae,Megasporaceae, Melanconidaceae, Melanommataceae, Melaspileaceae,Meliolaceae, Metacapnodiaceae, Microascaceae, Miltideaceae, Monascaceae,Monoblastiaceae, Montagnulaceae, Morchellaceae, Morosphaeriaceae,Mycoblastaceae, Mycocaliciaceae, Mycosphaerellaceae, Myeloconidaceae,Myriangiaceae, Myxotrichaceae, Nannizziopsidaceae, Nectriaceae,Nephromataceae, Niessliaceae, Nitschkiaceae, Obryzaceae,Ochrolechiaceae, Odontotremataceae, Onygenaceae, Ophiocordycipitaceae,Ophioparmaceae, Ophiostomataceae, Orbiliaceae, Pachyascaceae,Pannariaceae, Pannariaceae, Papulosaceae, Parmeliaceae, Parmulariaceae,Peltigeraceae, Peltulaceae, Pertusariaceae, Pezizaceae, Phacidiaceae,Phaeochoraceae, Phaeococcomycetaceae, Phaeosphaeriaceae,Phaeotrichaceae, Phaneromycetaceae, Phlyctidaceae, Phyllachoraceae,Physciaceae, Piedraiaceae, Pilocarpaceae, Placynthiaceae,Platystomaceae, Plectosphaerellaceae, Pleomassariaceae, Pleosporaceae,Pleurostomataceae, Porinaceae, Porpidiaceae, Protothelenellaceae,Pseudoplagiostomataceae, Pseudovalsaceae, Psoraceae, Pycnoraceae,Pyrenulaceae, Pyronemataceae, Pyxidiophoraceae, Ramalinaceae,Requienellaceae, Reticulascaceae, Rhizinaceae, Rhizocarpaceae,Rhynchostomataceae, Rhytismataceae, Roccellaceae, Roccellographaceae,Ropalosporaceae, Roussoellaceae, Rutstroemiaceae, Sagiolechiaceae,Salsugineaceae, Sarcoscyphaceae, Sarcosomataceae, Sarrameanaceae,Schaereriaceae, Schizoparmaceae, Schizoparmeaceae, Sclerotiniaceae,Scoliciosporaceae, Scortechiniaceae, Shiraiaceae, Sordariaceae,Spathulosporaceae, Sphaerophoraceae, Sphinctrinaceae, Sporastatiaceae,Sporormiaceae, Stereocaulaceae, Stictidaceae, Strigulaceae,Sydowiellaceae, Sympoventuriaceae, Teichosporaceae, Teloschistaceae,Teratosphaeriaceae, Testudinaceae, Tetraplosphaeriaceae, Thelebolaceae,Thelenellaceae, Thelocarpaceae, Thermoascaceae, Thyridariaceae,Thyridiaceae, Thyridiaceae, Togniniaceae, Trapeliaceae,Trematosphaeriaceae, Trichocomaceae, Trichomeriaceae,Trichosphaeriaceae, Tuberaceae, Tubeufiaceae, Umbilicariaceae,Vahliellaceae, Valsaceae, Venturiaceae, Verrucariaceae, Vezdaeaceae,Vialaeaceae, Vibrisseaceae, Xanthopyreniaceae, Xylariaceae,Xylonomycetaceae, and Zopfiaceae.

In some embodiments, the composition comprises anagriculturally-acceptable carrier and at least one spore-forming,filamentous bacterial endophyte of phylum Actinobacteria. In someembodiments, the at least one endophyte is a bacteria of orderactinomycetales. In some embodiments, the at least one endophyte isselected from one of the following families: Actinomycetaceae,Actinopolysporineae, Catenulisporineae, Corynebacterineae, Frankineae,Glycomycineae, Kineosporiineae, Micrococcineae, Micromonosporineae,Propionibacterineae, Pseudonocardineae, Streptomycineae, andStreptosporangineae.

In some embodiments, the present disclosure provides a compositioncomprising a carrier and an endophyte of Paraconyothirium sp. straindeposited as IDAC 081111-03 or comprising a DNA sequence with at least97% identity to SEQ ID NO:5; an endophyte of Pseudeurotium sp. straindeposited as IDAC 081111-02 or comprising a DNA sequence with at least97% identity to SEQ ID NO:4; an endophyte of Penicillium sp. straindeposited as IDAC 081111-01 or comprising a DNA sequence with at least97% identity to SEQ ID NO:3; an endophyte of Cladosporium sp. straindeposited as IDAC 200312-06 or comprising a DNA sequence with at least97% identity to SEQ ID NO:1; an endophyte of Sarocladium sp. straindeposited as IDAC 200312-05 or comprising a DNA sequence with at least97% identity to SEQ ID NO:2; and/or an endophyte of Streptomyces sp.strain deposited as IDAC 081111-06 or comprising a DNA sequence with atleast 97% sequence identity to SEQ ID NO:6. In certain embodiments, theendophyte of Paraconyothirium sp. strain comprises a DNA sequence withat least 98% identity to SEQ ID NO:5; the endophyte of Pseudeurotium sp.strain comprises a DNA sequence with at least 98% identity to SEQ IDNO:4; the endophyte of Penicillium sp. strain comprises a DNA sequencewith at least 98% identity to SEQ ID NO:3; the endophyte of Cladosporiumsp. strain comprises a DNA sequence with at least 98% identity to SEQ IDNO:1; the endophyte of Sarocladium sp. strain comprises a DNA sequencewith at least 98% identity to SEQ ID NO:2; and the endophyte ofStreptomyces sp. strain comprises a DNA sequence with at least 98%sequence identity to SEQ ID NO:6. In certain embodiments, the endophyteof Paraconyothirium sp. strain comprises a DNA sequence with at least99% identity to SEQ ID NO:5; the endophyte of Pseudeurotium sp. straincomprises a DNA sequence with at least 99% identity to SEQ ID NO:4; theendophyte of Penicillium sp. strain comprises a DNA sequence with atleast 99% identity to SEQ ID NO:3; the endophyte of Cladosporium sp.strain comprises a DNA sequence with at least 99% identity to SEQ IDNO:1; the endophyte of Sarocladium sp. strain comprises a DNA sequencewith at least 99% identity to SEQ ID NO:2; and the endophyte ofStreptomyces sp. strain comprises a DNA sequence with at least 99%sequence identity to SEQ ID NO:6. In certain embodiments, the endophyteof Paraconyothirium sp. strain comprises a DNA sequence of SEQ ID NO:5;the endophyte of Pseudeurotium sp. strain comprises a DNA sequence ofSEQ ID NO:4; the endophyte of Penicillium sp. strain comprises a DNAsequence of SEQ ID NO:3; the endophyte of Cladosporium sp. straincomprises a DNA sequence of SEQ ID NO:1; the endophyte of Sarocladiumsp. strain comprises a DNA sequence of SEQ ID NO:2; and the endophyte ofStreptomyces sp. strain comprises a DNA sequence of SEQ ID NO:6.

In some embodiments, the present disclosure provides a syntheticpreparation comprising an agricultural plant seed and a compositioncomprising endophytes capable of promoting germination and anagriculturally-acceptable carrier, wherein the synthetic preparation hasaltered gene expression in a plant grown from a seed inoculated withsaid composition, as compared to a control plant. In some embodiments,the composition is disposed on an exterior surface of an agriculturalseed in an amount effective to colonize the cortical cells of anagricultural plant grown from the seed and to alter the expression ofgenes involved in plant growth, genes associated with systemic acquiredresistance, or genes involved in protection from oxidative stress. Insome embodiments, these genes may be involved in phytohormoneproduction, for example in gibberellin (GA) biosynthesis or breakdown,abscisic acid (ABA) biosynthesis or breakdown, NO production orbreakdown, superoxide detoxification, or are positive or negativeregulators of these pathways. In other embodiments, the genes associatedwith systemic acquired resistance are redox-regulated transcriptionfactors. In still other embodiments, the redox-regulated transcriptionfactors belong to the MYB family of genes. In some embodiments, the genewith altered expression is selected from the group consisting of P5CS,SOD, MnSOD, GA3-oxidase 2, 14-3-3, NCED2, ABA8′OH1, RSG, KAO, Myb1 andMyb2. In some embodiments, the change in gene expression can be at least1%, for example at least 2%, at least 3%, at least 4%, at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 75%, at least 100%, or more, when compared with acontrol agricultural seed or plant. In some embodiments, saidcomposition is disposed on an exterior surface of an agricultural seedin an amount effective to colonize at least 0.1%, at least 1%, at least2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, orat least 80% of the cortical cells of an agricultural plant grown fromthe seed and to alter the expression of genes involved in plant growth,genes associated with systemic acquired resistance, or genes involved inprotection from oxidative stress.

In some embodiments, the present disclosure provides a compositioncomprising at least one endophyte capable of promoting germination andan agriculturally-acceptable carrier, wherein said composition isdisposed on an exterior surface of an agricultural seed in an amounteffective to cause a population of seeds inoculated with saidcomposition to have a faster dormancy breakdown, greater germinationrate, earlier germination, increased energy of germination, greater rateof germination, greater uniformity of germination, including greateruniformity of rate of germination and greater uniformity of timing ofgermination, and/or increased energy of germination as compared to apopulation of control seeds. In some embodiments, the composition isdisposed on the surface or within a tissue of an agricultural seed orseedling in an amount effective to cause a population of seedsinoculated with said composition to reach 50% germination faster than apopulation of control seeds or to cause increased NO accumulation in aplant grown from a seed inoculated with said composition, as compared toa control plant. In other embodiments, the composition is disposed on anexterior surface of an agricultural seed an in an amount effective tocause altered levels of phytohormones to be produced in an agriculturalplant grown from the seed, as compared to a control agricultural plant.In some embodiments, the phytohormones that are altered aregibberellins, abscisic acid, or cytokinins. In further embodiments, thegibberellins may be gibberellin 1, 19, 44 or 53. In still furtherembodiments, the cytokinin may be zeatin. For all these altered traits(a faster dormancy breakdown, greater germination rate, earliergermination, increased energy of germination, greater rate ofgermination, greater uniformity of germination, including greateruniformity of rate of germination and greater uniformity of timing ofgermination, increased energy of germination, 50% germination, increasedNO accumulation, and altered levels of phytohormones), the change can beat least 1%, for example at least 2%, at least 3%, at least 4%, at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 75%, at least 100%, or more, when comparedwith a control agricultural seed or plant.

In some embodiments, the present disclosure provides a compositioncomprising at least one endophyte and a carrier, wherein saidcomposition is capable of colonizing at least 0.1%, at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least20%, at least 30%, at least 40%, at least 50% at least 60%, at least70%, or at least 80% of cortex cells of a plant grown from a seedinoculated with said composition and wherein said plant has an improvedtrait as compared to a control plant. In certain embodiments, the plantgrown from seed inoculated with the composition has an improved traitselected from the group consisting of increased yield, faster seedlingestablishment, faster growth, increased drought tolerance, increasedheat tolerance, increased cold tolerance, increased salt tolerance,increased tolerance to Fusarium infection, increased biomass, increasedroot length, increased fresh weight of seedlings, increased plantvigour, nitrogen stress tolerance, enhanced Rhizobium activity, enhancednodulation frequency and early flowering time compared to a controlplant.

In another embodiment, the synthetic preparations and compositionsdescribed herein comprise two or more (e.g., 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 ormore, 20 or more, 25 or more, or greater than 25) different endophytescapable of promoting germination, e.g., obtained from different familiesor different genera of fungi or bacteria, or from the same genera butdifferent species of fungi or bacteria. In embodiments in which two ormore endophytes capable of promoting germination are used, each of theendophytes capable of promoting germination can have differentproperties or activities, confer different beneficial traits, orcolonize different parts of a plant (e.g., leaves, stems, flowers,fruits, seeds, or roots). For example, one endophyte capable ofpromoting germination can colonize a first tissue and a second endophytecapable of promoting germination can colonize a tissue that differs fromthe first tissue. Alternatively, each of the endophytes capable ofpromoting germination can have similar properties or activities, confersimilar beneficial traits, or colonize different parts of a plant.

The synthetic combination or preparation of the present inventioncontemplates the presence of an endophyte on the surface of the seed ofthe first plant. In one embodiment, the seed of the first plant iscoated with at least 10 CFU of the endophyte per seed, for example, atleast 20 CFU, at least 50 CFU, at least 100 CFU, at least 200 CFU, atleast 300 CFU, at least 500 CFU, at least 1,000 CFU, at least 3,000 CFU,at least 10,000 CFU, or at least 30,000 CFU or more per seed. In anotherembodiment, the seed is coated with at least 10, for example, at least20, at least 50, at least 100, at least 200, at least 300, at least 500,at least 1,000, at least 3,000, at least 10,000, at least 30,000, atleast 100,000, at least 300,000, at least 1,000,000 or more of theendophyte as determined by the number of copies of a particularendophyte gene detected, for example, by quantitative PCR.

Further provided herein is a seed inoculated with any of thecompositions described herein. In one embodiment, the seed is inoculatedby soil-based inoculation. In another embodiment, the seed is coatedwith an endophyte or culture thereof. In yet another embodiment, theseed is sprayed, injected, inoculated, grafted, coated or treated withthe endophyte or culture thereof. In an embodiment, the seed is plantednear an endophyte. In one embodiment, the seed planted near theendophyte is about 4 cm away from the endophyte.

In another aspect, the invention provides a population of at least 10synthetic preparations, each synthetic preparation comprising anagricultural plant seed and a composition comprising at least oneendophyte capable of promoting germination and anagriculturally-acceptable carrier, where the population is comprisedwithin a packaging material. The packaging material can be selected froma bag, box, bin, envelope, carton, or container. In an embodiment, thesynthetic preparation can be disposed within a package and is shelfstable. In another embodiment, the invention features an agriculturalproduct that includes a predetermined number of seeds or a predeterminedweight of seeds. In an embodiment, the bag or container contains atleast 1000 seeds, wherein the packaging material optionally comprises adessicant, and wherein the synthetic preparation optionally comprises ananti-fungal agent.

In yet another aspect, the invention features an article of manufacturethat includes packaging material; one or more plant seeds within thepackaging material, and at least one species of endophytes capable ofpromoting germination associated with the seeds. The article can includetwo or more species of endophytes capable of promoting germination.

In another aspect, the invention features an agricultural product thatincludes a predetermined number of seeds or a predetermined weight ofseeds. In an embodiment, the bag or container contains at least 1000seeds of a synthetic preparation produced by the step of inoculating aplurality of plant seeds with a formulation comprising a fungal orbacterial population at a concentration of at least 1 CFU peragricultural plant seed, wherein at least 10% of the CFUs present in theformulation are one or more endophytes capable of promoting germination,under conditions such that the formulation is associated with thesurface of the seeds in a manner effective for the endophytes capable ofpromoting germination to confer a benefit to the seeds or to a cropcomprising a plurality of agricultural plants produced from the seeds.The endophytes capable of promoting germination can be present in aconcentration of from about 10² to about 10⁵ CFU/ml or from about 10⁵ toabout 10⁸ CFU/seed. The formulation can be a liquid and the fungal orbacterial concentration can be from about 10³ to about 10¹¹ CFU/ml. Theformulation can be a gel or powder and the fungal or bacterialconcentration can be from about 10³ to about 10¹¹ CFU/gm.

In some cases, the endophytic microbe can be modified. For example, theendophytic microbe can be genetically modified by introduction of atransgene that stably integrates into its genome. In another embodiment,the endophytic microbe can be modified to harbor a plasmid or episomecontaining a transgene. In still another embodiment, the microbe can bemodified by repeated passaging under selective conditions.

The microbe can be modified to exhibit altered characteristics. In oneembodiment, the endophytic microbe is modified to exhibit increasedcompatibility with chemicals commonly used in agriculture. Agriculturalplants are often treated with a vast array of agrichemicals, includingfungicides, biocides (anti-bacterial and anti-fungal agents),herbicides, insecticides, nematicides, rodenticides, fertilizers, andother agents. Many such agents can affect the ability of an endophyticmicrobe to grow, divide, and/or otherwise confer beneficial traits tothe plant.

In some cases, it can be important for the microbe to be compatible withagrichemicals, particularly those with fungicidal or antibacterialproperties, in order to persist in the plant although, as mentionedearlier, there are many such fungicidal or antibacterial agents that donot penetrate the plant, at least at a concentration sufficient tointerfere with the microbe. Therefore, where a systemic fungicide orantibacterial agent is used in the plant, compatibility of the microbeto be inoculated with such agents will be an important criterion.

In one embodiment, spontaneous isolates of microbes that are compatiblewith agrichemicals can be used to inoculate the plants according to themethods described herein. For example, fungal microbes which arecompatible with agriculturally employed fungicides can be isolated byplating a culture of the microbes on a petri dish containing aneffective concentration of the fungicide, and isolating colonies of themicrobe that are compatible with the fungicide. In another embodiment, amicrobe that is compatible with a fungicide is used for the methodsdescribed herein. For example, the endophyte can be compatible with atleast one of the fungicides selected from the group consisting of:2-(thiocyanatomethylthio)-benzothiazole, 2-phenylphenol,8-hydroxyquinoline sulfate, ametoctradin, amisulbrom, antimycin,Ampelomyces quisqualis, azaconazole, azoxystrobin, Bacillus subtilis,benalaxyl, benomyl, benthiavalicarb-isopropyl,benzylaminobenzene-sulfonate (BABS) salt, bicarbonates, biphenyl,bismerthiazol, bitertanol, bixafen, blasticidin-S, borax, Bordeauxmixture, boscalid, bromuconazole, bupirimate, calcium polysulfide,captafol, captan, carbendazim, carboxin, carpropamid, carvone,chloroneb, chlorothalonil, chlozolinate, Coniothyrium minitans, copperhydroxide, copper octanoate, copper oxychloride, copper sulfate, coppersulfate (tribasic), cuprous oxide, cyazofamid, cyflufenamid, cymoxanil,cyproconazole, cyprodinil, dazomet, debacarb, diammoniumethylenebis-(dithiocarbamate), dichlofluanid, dichlorophen, diclocymet,diclomezine, dichloran, diethofencarb, difenoconazole, difenzoquat ion,diflumetorim, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M,dinobuton, dinocap, diphenylamine, dithianon, dodemorph, dodemorphacetate, dodine, dodine free base, edifenphos, enestrobin,epoxiconazole, ethaboxam, ethoxyquin, etridiazole, famoxadone,fenamidone, fenarimol, fenbuconazole, fenfuram, fenhexamid, fenoxanil,fenpiclonil, fenpropidin, fenpropimorph, fentin, fentin acetate, fentinhydroxide, ferbam, ferimzone, fluazinam, fludioxonil, flumorph,fluopicolide, fluopyram, fluoroimide, fluoxastrobin, fluquinconazole,flusilazole, flusulfamide, flutianil, flutolanil, flutriafol,fluxapyroxad, folpet, formaldehyde, fosetyl, fosetyl-aluminium,fuberidazole, furalaxyl, furametpyr, guazatine, guazatine acetates,GY-81, hexachlorobenzene, hexaconazole, hymexazol, imazalil, imazalilsulfate, imibenconazole, iminoctadine, iminoctadine triacetate,iminoctadine tris(albesilate), ipconazole, iprobenfos, iprodione,iprovalicarb, isoprothiolane, isopyrazam, isotianil, kasugamycin,kasugamycin hydrochloride hydrate, kresoxim-methyl, mancopper, mancozeb,mandipropamid, maneb, mepanipyrim, mepronil, mercuric chloride, mercuricoxide, mercurous chloride, metalaxyl, mefenoxam, metalaxyl-M, metam,metam-ammonium, metam-potassium, metam-sodium, metconazole,methasulfocarb, methyl iodide, methyl isothiocyanate, metiram,metominostrobin, metrafenone, mildiomycin, myclobutanil, nabam,nitrothal-isopropyl, nuarimol, octhilinone, ofurace, oleic acid (fattyacids), orysastrobin, oxadixyl, oxine-copper, oxpoconazole fumarate,oxycarboxin, pefurazoate, penconazole, pencycuron, penflufen,pentachlorophenol, pentachlorophenyl laurate, penthiopyrad,phenylmercury acetate, phosphonic acid, phthalide, picoxystrobin,polyoxin B, polyoxins, polyoxorim, potassium bicarbonate, potassiumhydroxyquinoline sulfate, probenazole, prochloraz, procymidone,propamocarb, propamocarb hydrochloride, propiconazole, propineb,proquinazid, prothioconazole, pyraclostrobin, pyrametostrobin,pyraoxystrobin, pyrazophos, pyribencarb, pyributicarb, pyrifenox,pyrimethanil, pyroquilon, quinoclamine, quinoxyfen, quintozene,Reynoutria sachalinensis extract, sedaxane, silthiofam, simeconazole,sodium 2-phenylphenoxide, sodium bicarbonate, sodiumpentachlorophenoxide, spiroxamine, sulfur, SYP-Z071, SYP-Z048, tar oils,tebuconazole, tebufloquin, tecnazene, tetraconazole, thiabendazole,thifluzamide, thiophanate-methyl, thiram, tiadinil, tolclofos-methyl,tolylfluanid, triadimefon, triadimenol, triazoxide, tricyclazole,tridemorph, trifloxystrobin, triflumizole, triforine, triticonazole,validamycin, valifenalate, valiphenal, vinclozolin, zineb, ziram,zoxamide, Candida oleophila, Fusarium oxysporum, Gliocladium spp.,Phlebiopsis gigantea, Streptomyces griseoviridis, Trichoderma spp.,(RS)—N-(3,5-dichlorophenyl)-2-(methoxymethyl)-succinimide,1,2-dichloropropane, 1,3-dichloro-1,1,3,3-tetrafluoroacetone hydrate,1-chloro-2,4-dinitronaphthalene, 1-chloro-2-nitropropane,2-(2-heptadecyl-2-imidazolin-1-yl)ethanol,2,3-dihydro-5-phenyl-1,4-dithi-ine 1,1,4,4-tetraoxide,2-methoxyethylmercury acetate, 2-methoxyethylmercury chloride,2-methoxyethylmercury silicate, 3-(4-chlorophenyl)-5-methylrhodanine,4-(2-nitroprop-1-enyl)phenyl thiocyanateme, ampropylfos, anilazine,azithiram, barium polysulfide, Bayer 32394, benodanil, benquinox,bentaluron, benzamacril; benzamacril-isobutyl, benzamorf, binapacryl,bis(methylmercury) sulfate, bis(tributyltin) oxide, buthiobate, cadmiumcalcium copper zinc chromate sulfate, carbamorph, CECA, chlobenthiazone,chloraniformethan, chlorfenazole, chlorquinox, climbazole, cyclafuramid,cypendazole, cyprofuram, decafentin, dichlone, dichlozoline,diclobutrazol, dimethirimol, dinocton, dinosulfon, dinoterbon,dipyrithione, ditalimfos, dodicin, drazoxolon, EBP, ESBP, etaconazole,etem, ethirim, fenaminosulf, fenapanil, fenitropan, 5-fluorocytosine andprofungicides thereof, fluotrimazole, furcarbanil, furconazole,furconazole-cis, furmecyclox, furophanate, glyodine, griseofulvin,halacrinate, Hercules 3944, hexylthiofos, ICIA0858, isopamphos,isovaledione, mebenil, mecarbinzid, metazoxolon, methfuroxam,methylmercury dicyandiamide, metsulfovax, milneb, mucochloric anhydride,myclozolin, N-3,5-dichlorophenyl-succinimide,N-3-nitrophenylitaconimide, natamycin,N-ethylmercurio-4-toluenesulfonanilide, nickelbis(dimethyldithiocarbamate), OCH, phenylmercurydimethyldithiocarbamate, phenylmercury nitrate, phosdiphen, picolinamideUK-2A and derivatives thereof, prothiocarb; prothiocarb hydrochloride,pyracarbolid, pyridinitril, pyroxychlor, pyroxyfur, quinacetol;quinacetol sulfate, quinazamid, quinconazole, rabenzazole,salicylanilide, SSF-109, sultropen, tecoram, thiadifluor, thicyofen,thiochlorfenphim, thiophanate, thioquinox, tioxymid, triamiphos,triarimol, triazbutil, trichlamide, urbacid, XRD-563, and zarilamide,IK-1140

In still another embodiment, an endophyte that is compatible with anantibacterial compound is used for the methods described herein. Forexample, the endophyte can be compatible with at least one of theantibiotics selected from the group consisting of: Amikacin, Gentamicin,Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Spectinomycin,Geldanamycin, Herbimycin, Rifaximin, streptomycin, Loracarbef,Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil,Cefazolin, Cefalotin or Cefalothin, Cefalexin, Cefaclor, Cefamandole,Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren,Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten,Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole,Teicoplanin, Vancomycin, Telavancin, Clindamycin, Lincomycin,Daptomycin, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin,Roxithromycin, Troleandomycin, Telithromycin, Spiramycin, Aztreonam,Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid,Torezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin,Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin,Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin,Penicillin G, Temocillin, Ticarcillin, Amoxicillin/clavulanate,Ampicillin/sulbactam, Piperacillin/tazobactam, Ticarcillin/clavulanate,Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin,Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid,Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin,Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silversulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole,Sulfanilimide (archaic), Sulfasalazine, Sulfisoxazole,Trimethoprim-Sulfamethoxazole (Co-trimoxazole) (TMP-SMX),Sulfonamidochrysoidine (archaic), Demeclocycline, Doxycycline,Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone,Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid,Pyrazinamide, Rifampicin (Rifampin in US), Rifabutin, Rifapentine,Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid,Metronidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin,Thiamphenicol, Tigecycline, Tinidazole, and Trimethoprim. Fungicidecompatible microbes can also be isolated by selection on liquid medium.The culture of microbes can be plated on petri dishes without any formsof mutagenesis; alternatively, the microbes can be mutagenized using anymeans known in the art. For example, microbial cultures can be exposedto UV light, gamma-irradiation, or a chemical mutagen such asethylmethanesulfonate (EMS) prior to selection on fungicide containingmedia. Finally, where the mechanism of action of a particular fungicideis known, the target gene can be specifically mutated (either by genedeletion, gene replacement, site-directed mutagenesis, etc.) to generatea microbe that is resilient against that particular fungicide. It isnoted that the above-described methods can be used to isolate fungi thatare compatible with both fungistatic and fungicidal compounds.

It will also be appreciated by one skilled in the art that a plant maybe exposed to multiple types of fungicides or antibacterial compounds,either simultaneously or in succession, for example at different stagesof plant growth. Where the target plant is likely to be exposed tomultiple fungicidal and/or antibacterial agents, a microbe that iscompatible with many or all of these agrichemicals can be used toinoculate the plant. A microbe that is compatible with severalfungicidal agents can be isolated, for example, by serial selection. Amicrobe that is compatible with the first fungicidal agent is isolatedas described above (with or without prior mutagenesis). A culture of theresulting microbe can then be selected for the ability to grow on liquidor solid media containing the second antifungal compound (again, with orwithout prior mutagenesis). Colonies isolated from the second selectionare then tested to confirm its compatibility to both antifungalcompounds.

Likewise, bacterial microbes that are compatible to biocides (includingherbicides such as glyphosate or antibacterial compounds, whetherbacteriostatic or bactericidal) that are agriculturally employed can beisolated using methods similar to those described for isolatingfungicide compatible microbes. In one embodiment, mutagenesis of themicrobial population can be performed prior to selection with anantibacterial agent. In another embodiment, selection is performed onthe microbial population without prior mutagenesis. In still anotherembodiment, serial selection is performed on a microbe: the microbe isfirst selected for compatibility to a first antibacterial agent. Theisolated compatible microbe is then cultured and selected forcompatibility to the second antibacterial agent. Any colony thusisolated is tested for compatibility to each, or both antibacterialagents to confirm compatibility with these two agents.

The selection process described above can be repeated to identifyisolates of the microbe that are compatible with a multitude ofantifungal or antibacterial agents. Candidate isolates can be tested toensure that the selection for agrichemical compatibility did not resultin loss of a desired microbial bioactivity. Isolates of the microbe thatare compatible with commonly employed fungicides can be selected asdescribed above. The resulting compatible microbe can be compared withthe parental microbe on plants in its ability to promote germination.

Methods

Further provided herein are methods of enhancing seed vitality, planthealth and/or yield comprising inoculating a seed with an endophyte orculture disclosed herein or a combination or mixture thereof or with acomposition disclosed herein. In some embodiments, a first generationplant is cultivated from the seed.

In one aspect, the invention provides a method of altering a trait in anagricultural plant seed or an agricultural plant grown from said seed,said method comprising inoculating said seed with a compositioncomprising endophytes capable of promoting germination and anagriculturally-acceptable carrier, wherein the endophyte replicateswithin at least one plant tissue and colonizes the cortical cells ofsaid plant. In one embodiment, the endophyte capable of promotinggermination is a coleorhiza-activating endophyte, and the seed is amonocot seed. In another embodiment, the endophyte capable of promotinggermination is heterologous to the seed.

In some embodiments, the endophytes are a selected from the groupconsisting of a spore-forming endophyte, a facultative endophyte, afilamentous endophyte, and an endophyte capable of living within anotherendophyte. In some embodiments, the endophyte is capable of formingcertain structures in the plant, where the structures are selected fromthe group consisting of hyphal coils, Hartig-like nets, microvesicles,micro-arbuscules, hyphal knots, and symbiosomes. In some embodiments,the endophyte is in the form of at least one of conidia, chlamydospore,and mycelia. In other embodiments, the fungus or bacteria is capable ofbeing part of a plant-fungus symbiotic system or plant-bacteriasymbiotic system that produces altered levels of phytohormones oranti-oxidants, as compared to a plant that is not in symbiosis. In otherembodiments, the plant-fungus symbiotic system or plant-bacteriumsymbiotic system has anti-aging and/or anti-senescence effects, ascompared to a plant or plant organ that is not in symbiosis. In otherembodiments, the plant-fungus symbiotic system or plant-bacteriasymbiotic system has increased protection against pathogens, as comparedto a plant that is not in symbiosis. In other aspects, the endophytecolonizes at least 0.1%, at least 1%, at least 2%, at least 3%, at least4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, or at least 80% of thecortical cells of said agricultural plant.

In yet another aspect, the invention provides a method of altering atrait in an agricultural plant seed or an agricultural plant grown fromsaid seed, said method comprising inoculating said seed with acomposition comprising endophytes capable of promoting germination andan agriculturally-acceptable carrier, wherein the altered trait is animproved functional trait selected from the group consisting ofincreased yield, faster seedling establishment, faster growth, increaseddrought tolerance, increased heat tolerance, increased cold tolerance,increased salt tolerance, increased tolerance to pests and diseases,increased biomass, increased root and/or shoot length or weight,increased fresh weight of seedlings, increased seed or fruit number,increased plant vigour, nitrogen stress tolerance, enhanced Rhizobiumactivity, enhanced nodulation frequency, early flowering time, or anycombination thereof. In some embodiments, the increased tolerance todisease is increased tolerance to Fusarium infection, increasedtolerance to Septoria infection, increased tolerance to Pucciniainfection. In some embodiments, yield is measured on a population ofplants grown in the field and is calculated via combine harvesting ormeasuring ear weight. In another aspect, the altered trait is a seedtrait selected from the group consisting a greater germination rate,faster dormancy breakdown, increased energy of germination, increasedseed germination vigor or increased seed vitality. In yet anotherembodiment, the altered trait is altered gene expression, wherein thegene is selected from the group consisting of a gene involved ingibberellin production, a gene involved in abscisic acid production, agene involved in plant growth, an acquired resistance gene, and a geneinvolved in protection from oxidative stress. In some embodiments, thegenes may be involved in phytohormone production. In some embodiments,the phytohormone is altered in the plant-fungus or plant-bacterialsymbiotic system. In some embodiments, the method further comprisesplanting the agricultural plant seed. In another embodiment, the methodfurther comprises selecting a plant seed or plant that has the alteredtrait. For all altered traits, the change can be at least 1%, forexample at least 2%, at least 3%, at least 4%, at least 5%, at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 75%, at least 100%, or more, when compared with a controlagricultural seed or plant.

In one embodiment, the invention provides methods of improving the 50%germination rate of a population of seeds comprising inoculating saidpopulation of seeds with a composition as described herein. In oneembodiment, the method is a method of improving the 50% germination rateof a population of seeds and of improving a trait in plants grown fromthe seeds, comprising inoculating said population of seeds with acomposition as described herein. In one embodiment, the improved traitis selected from the group consisting of increased yield, fasterseedling establishment, faster growth, increased drought tolerance,increased heat tolerance, increased cold tolerance, increased salttolerance, increased tolerance to pests and diseases, increased biomass,increased root and/or shoot length or weight, increased fresh weight ofseedlings, increased seed or fruit number, increased plant vigour,nitrogen stress tolerance, enhanced Rhizobium activity, enhancednodulation frequency, early flowering time, or any combination thereof.In some embodiments, the increased tolerance to disease is increasedtolerance to Fusarium infection, increased tolerance to Septoriainfection, increased tolerance to Puccinia infection. In someembodiments, yield is measured on a population of plants grown in thefield and is calculated via combine harvesting or measuring ear weight.For all altered traits, the change can be at least 1%, for example atleast 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least75%, at least 100%, or more, when compared with a control agriculturalseed or plant. In some embodiments, the method further comprisesplanting the agricultural plant seed. In another embodiment, the methodfurther comprises selecting a plant seed or plant that has the alteredtrait.

In one embodiment, the method is a method of improving the 50%germination rate of a population of seeds and altering the geneexpression in a plant grown from the seeds, comprising inoculating saidpopulation of seeds with a composition as described herein. In someembodiments, the gene is altered in the plant-fungus or plant-bacterialsymbiotic system. In some embodiments, the gene with altered expressionis a gene involved in plant growth, an acquired resistance gene, and agene involved in protection from oxidative stress. In some embodiments,these genes may be involved in phytohormone production, such as thoseinvolved in GA biosynthesis or breakdown, abscisic acid (ABA)biosynthesis or breakdown, NO production or breakdown, superoxidedetoxification, or are positive or negative regulators of thesepathways. In other embodiments, the genes associated with systemicacquired resistance are redox-regulated transcription factors. In stillother embodiments, the redox-regulated transcription factors belong tothe MYB family of genes. In some embodiments, the gene with alteredexpression is selected from the group consisting of P5CS, SOD, MnSOD,GA3-oxidase 2, 14-3-3, NCED2, ABA8′OH1, RSG, KAO, Myb1 and Myb2. In someembodiments, the change in gene expression can be at least 1%, forexample at least 2%, at least 3%, at least 4%, at least 5%, at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 75%, at least 100%, or more, when compared with a controlagricultural seed or plant. In some embodiments, the method furthercomprises planting the agricultural plant seed. In another embodiment,the method further comprises selecting a plant seed or plant that hasthe altered trait.

In one embodiment, a method of improving the 50% germination rate of apopulation of seeds and providing at least 0.1%, at least 1%, at least2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, orat least 80% colonization in cortex cells of plants grown from the seedsis provided.

In one embodiment, methods of obtaining at least 0.1%, at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, or at least 80% endophyte colonization in the cortex cells of aplant and of improving a trait in the plant are disclosed, comprisinginoculating the seed of said plant with a composition as describedherein. In one embodiment, the improved trait is selected from the groupconsisting of increased yield, faster seedling establishment, fastergrowth, increased drought tolerance, increased heat tolerance, increasedcold tolerance, increased salt tolerance, increased tolerance to pestsand diseases, increased biomass, increased root and/or shoot length orweight, increased fresh weight of seedlings, increased seed or fruitnumber, increased plant vigour, nitrogen stress tolerance, enhancedRhizobium activity, enhanced nodulation frequency, early flowering time,or any combination thereof. In some embodiments, the increased toleranceto disease is increased tolerance to Fusarium infection, increasedtolerance to Septoria infection, increased tolerance to Pucciniainfection. In some embodiments, yield is measured on a population ofplants grown in the field and is calculated via combine harvesting ormeasuring ear weight. For all altered traits, the change can be at least1%, for example at least 2%, at least 3%, at least 4%, at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 75%, at least 100%, or more, when compared with acontrol agricultural seed or plant.

In one embodiment, the method is a method of increasing the germinationrate, speeding up dormancy breakdown, increasing the energy ofgermination, increasing the germination vigour, speeding up germination,increasing the energy of germination, producing greater uniformity ofgermination, including greater uniformity of rate of germination andgreater uniformity of timing of germination, or increasing the vitalityof a seed, comprising inoculating seeds or a population of seeds with acomposition as described herein. In some embodiments, the method furthercomprises planting the agricultural plant seed.

In one embodiment, the invention provides a method of releasing a seedfrom dormancy, said method comprising inoculating said seed with acomposition comprising endophytes capable of promoting germination andan agriculturally-acceptable carrier. In some embodiments, theendophytes capable of promoting germination are coleorhiza-activatingendophytes.

In one embodiment, the invention provides a method of improving the 50%germination rate of a population of seeds and increasing NO accumulationin a plant grown from the seeds, comprising inoculating seeds with acomposition as described herein. In some embodiments, the method furthercomprises planting the agricultural plant seed.

In another embodiment, a method of altering a trait in an agriculturalplant seed or an agricultural plant grown from said seed is disclosed,comprising obtaining a synthetic preparation comprising an agriculturalplant seed and a composition comprising endophytes capable of promotinggermination and an agriculturally-acceptable carrier and planting thesynthetic preparation. In some embodiments, the method further comprisesplanting the agricultural plant seed. In another embodiment, the methodfurther comprises selecting a plant seed or plant that has the alteredtrait.

In another embodiment, the invention provides a method for treatingseeds comprising contacting the surface of an agricultural plant seedwith a formulation comprising a microbial population that comprises anendophyte capable of promoting germination that is heterologous to theseed, wherein the endophyte capable of promoting germination is presentin the formulation in an amount effective to alter the level of at leastone gene within the seed, seedlings derived from the seed oragricultural plants derived from the seed. In some embodiments, the genewith altered expression is a gene involved in phytohormone production,an acquired resistance gene, and a gene involved in protection fromoxidative stress. In some embodiments, these genes are those involved inGA biosynthesis or breakdown, abscisic acid (ABA) biosynthesis orbreakdown, NO production or breakdown, superoxide detoxification, or arepositive or negative regulators of these pathways. In other embodiments,the genes associated with systemic acquired resistance areredox-regulated transcription factors. In still other embodiments, theredox-regulated transcription factors belong to the MYB family of genes.In some embodiments, the gene with altered expression is selected fromthe group consisting of P5CS, SOD, MnSOD, GA3-oxidase 2, 14-3-3, NCED2,ABA8′OH1, RSG, KAO, Myb1 and Myb2.

In another embodiment, the invention provides a method for treatingseeds comprising contacting the surface of an agricultural plant seedwith a formulation comprising a microbial population that comprises anendophyte capable of promoting germination that is heterologous to theseed, wherein the endophyte capable of promoting germination is presentin the formulation in an amount effective to alter the level of at leastone phytohormone within the seed, seedlings derived from the seed oragricultural plants derived from the seed. In some embodiments, thephytohormones that are altered are gibberellins, abscisic acid, orcytokinins. In further embodiments, the gibberellins may be gibberellin1, 19, 44 or 53. In still further embodiments, the cytokinin may bezeatin. For these altered phytohormone levels, the change can be atleast 1%, for example at least 2%, at least 3%, at least 4%, at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 75%, at least 100%, or more, when comparedwith a control agricultural seed or plant.

In another embodiment, the invention provides a method for treatingseeds comprising contacting the surface of an agricultural plant seedwith a formulation comprising a microbial population that comprises anendophyte capable of promoting germination that is heterologous to theseed, wherein the endophyte capable of promoting germination is presentin the formulation in an amount effective to improve a trait in the seedor a plant grown from the seed. In some embodiments, the improved traitis selected from the group consisting of increased yield, fasterseedling establishment, faster growth, increased drought tolerance,increased heat tolerance, increased cold tolerance, increased salttolerance, increased tolerance to pests and diseases, increased biomass,increased root and/or shoot length or weight, increased fresh weight ofseedlings, increased seed or fruit number, increased plant vigour,nitrogen stress tolerance, enhanced Rhizobium activity, enhancednodulation frequency, early flowering time, or any combination thereof.In some embodiments, the increased tolerance to disease is increasedtolerance to Fusarium infection, increased tolerance to Septoriainfection, increased tolerance to Puccinia infection. In someembodiments, yield is measured on a population of plants grown in thefield and is calculated via combine harvesting or measuring ear weight.For all altered traits, the change can be at least 1%, for example atleast 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least75%, at least 100%, or more, when compared with a control agriculturalseed or plant. In some embodiments, the method further comprisesplanting the agricultural plant seed. In another embodiment, the methodfurther comprises selecting a plant seed or plant that has the alteredtrait.

In another aspect, there is provided a method of improving plant healthand/or plant yield comprising treating plant propagation material or aplant with a composition disclosed herein; and cultivating the plantpropagation material into a first generation plant or allowing the plantto grow.

In another embodiment, the methods reduce the effects of stress, such asheat, drought and/or biotic stress.

In an embodiment, the methods enhance landscape development andremediation.

Accordingly, in one embodiment, there is provided a method ofphytoremediation or phytoreclamation of a contaminated site comprisingtreating plant propagation material or a plant with a compositiondisclosed herein, and cultivating the plant propagation material into afirst generation plant or allowing the plant to grow; therebyremediating or reclaiming the site.

In one embodiment, the site is soil, such as at a landfill. In anembodiment, the substances, wastes or hazardous materials comprisehydrocarbons, petroleum or other chemicals, salts, or metals, such aslead, cadmium or radioisotopes.

Formulations/Seed coating compositions

The purified endophytes described herein can be formulated using anagriculturally compatible carrier. The formulation useful for theseembodiments generally typically include at least one member selectedfrom the group consisting of a tackifier, a microbial stabilizer, afungicide, an antibacterial agent, an herbicide, a nematicide, aninsecticide, a plant growth regulator, a rodenticide, a dessicant, and anutrient.

In some cases, the purified bacterial or fungal population is mixed withan agriculturally compatible carrier. The carrier can be a solid carrieror liquid carrier, and in various forms including microspheres, powders,emulsions and the like. The carrier may be any one or more of a numberof carriers that confer a variety of properties, such as increasedstability, wettability, or dispersability. Wetting agents such asnatural or synthetic surfactants, which can be nonionic or ionicsurfactants, or a combination thereof can be included in a compositionof the invention. Water-in-oil emulsions can also be used to formulate acomposition that includes the purified bacterial or fungal population(see, for example, U.S. Pat. No. 7,485,451, which is incorporated hereinby reference in its entirety). Suitable formulations that may beprepared include wettable powders, granules, gels, agar strips orpellets, thickeners, and the like, microencapsulated particles, and thelike, liquids such as aqueous flowables, aqueous suspensions,water-in-oil emulsions, etc. The formulation may include grain or legumeproducts, for example, ground grain or beans, broth or flour derivedfrom grain or beans, starch, sugar, or oil.

In some embodiments, the agricultural carrier may be soil or a plantgrowth medium. Other agricultural carriers that may be used includewater, fertilizers, plant-based oils, humectants, or combinationsthereof. Alternatively, the agricultural carrier may be a solid, such asdiatomaceous earth, loam, silica, alginate, clay, bentonite,vermiculite, seed cases, other plant and animal products, orcombinations, including granules, pellets, or suspensions. Mixtures ofany of the aforementioned ingredients are also contemplated as carriers,such as but not limited to, pesta (flour and kaolin clay), agar orflour-based pellets in loam, sand, or clay, etc. Formulations mayinclude food sources for the cultured organisms, such as barley, rice,or other biological materials such as seed, plant parts, sugar canebagasse, hulls or stalks from grain processing, ground plant material orwood from building site refuse, sawdust or small fibers from recyclingof paper, fabric, or wood. Other suitable formulations will be known tothose skilled in the art.

In one embodiment, the formulation can include a tackifier or adherent.Such agents are useful for combining the bacterial or fungal populationof the invention with carriers that can contain other compounds (e.g.,control agents that are not biologic), to yield a coating composition.Such compositions help create coatings around the plant or seed tomaintain contact between the microbe and other agents with the plant orplant part. In one embodiment, adherents are selected from the groupconsisting of: alginate, gums, starches, lecithins, formononetin,polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinylacetate, cephalins, Gum Arabic, Xanthan Gum, Mineral Oil, PolyethyleneGlycol (PEG), Polyvinyl pyrrolidone (PVP), Arabino-galactan, MethylCellulose, PEG 400, Chitosan, Polyacrylamide, Polyacrylate,Polyacrylonitrile, Glycerol, Triethylene glycol, Vinyl Acetate, GellanGum, Polystyrene, Polyvinyl, Carboxymethyl cellulose, Gum Ghatti, andpolyoxyethylene-polyoxybutylene block copolymers. Other examples ofadherent compositions that can be used in the synthetic preparationinclude those described in EP 0818135, CA 1229497, WO 2013090628, EP0192342, WO 2008103422 and CA 1041788, each of which is incorporatedherein by reference in its entirety.

The formulation can also contain a surfactant. Non-limiting examples ofsurfactants include nitrogen-surfactant blends such as Prefer 28(Cenex), Surf-N(US), Inhance (Brandt), P-28 (Wilfarm) and Patrol(Helena); esterified seed oils include Sun-It II (AmCy), MSO (UAP),Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel); andorgano-silicone surfactants include Silwet L77 (UAP), Silikin (Terra),Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis) andCentury (Precision). In one embodiment, the surfactant is present at aconcentration of between 0.01% v/v to 10% v/v. In another embodiment,the surfactant is present at a concentration of between 0.1% v/v to 1%v/v.

In certain cases, the formulation includes a microbial stabilizer. Suchan agent can include a desiccant. As used herein, a “desiccant” caninclude any compound or mixture of compounds that can be classified as adesiccant regardless of whether the compound or compounds are used insuch concentrations that they in fact have a desiccating effect on theliquid inoculant. Such desiccants are ideally compatible with thebacterial or fungal population used, and should promote the ability ofthe microbial population to survive application on the seeds and tosurvive desiccation. Examples of suitable desiccants include one or moreof trehalose, sucrose, glycerol, and Methylene glycol. Other suitabledesiccants include, but are not limited to, non-reducing sugars andsugar alcohols (e.g., mannitol or sorbitol). The amount of desiccantintroduced into the formulation can range from about 5% to about 50% byweight/volume, for example, between about 10% to about 40%, betweenabout 15% and about 35%, or between about 20% and about 30%.

In some cases, it is advantageous for the formulation to contain agentssuch as a fungicide, an antibacterial agent, an herbicide, a nematicide,an insecticide, a plant growth regulator, a rodenticide, or a nutrient.Such agents are ideally compatible with the agricultural seed orseedling onto which the formulation is applied (e.g., it should not bedeleterious to the growth or health of the plant). Furthermore, theagent is ideally one that does not cause safety concerns for human,animal or industrial use (e.g., no safety issues, or the compound issufficiently labile that the commodity plant product derived from theplant contains negligible amounts of the compound).

In the liquid form, for example, solutions or suspensions, the bacterialor fungal endophytic populations of the present invention can be mixedor suspended in water or in aqueous solutions. Suitable liquid diluentsor carriers include water, aqueous solutions, petroleum distillates, orother liquid carriers.

Solid compositions can be prepared by dispersing the bacterial or fungalendophytic populations of the invention in and on an appropriatelydivided solid carrier, such as peat, wheat, bran, vermiculite, clay,talc, bentonite, diatomaceous earth, fuller's earth, pasteurized soil,and the like. When such formulations are used as wettable powders,biologically compatible dispersing agents such as non-ionic, anionic,amphoteric, or cationic dispersing and emulsifying agents can be used.

The solid carriers used upon formulation include, for example, mineralcarriers such as kaolin clay, pyrophyllite, bentonite, montmorillonite,diatomaceous earth, acid white soil, vermiculite, and pearlite, andinorganic salts such as ammonium sulfate, ammonium phosphate, ammoniumnitrate, urea, ammonium chloride, and calcium carbonate. Also, organicfine powders such as wheat flour, wheat bran, and rice bran may be used.The liquid carriers include vegetable oils such as soybean oil andcottonseed oil, glycerol, ethylene glycol, polyethylene glycol,propylene glycol, polypropylene glycol, etc.

In one particular embodiment, the formulation is ideally suited forcoating of the endophytic microbial population onto seeds. The bacterialor fungal endophytic populations described in the present invention arecapable of conferring many fitness benefits to the host plants. Theability to confer such benefits by coating the bacterial or fungalpopulations on the surface of seeds has many potential advantages,particularly when used in a commercial (agricultural) scale.

The bacterial or fungal endophytic populations herein can be combinedwith one or more of the agents described above to yield a formulationsuitable for combining with an agricultural seed or seedling. Thebacterial or fungal population can be obtained from growth in culture,for example, using a synthetic growth medium. In addition, the microbecan be cultured on solid media, for example on petri dishes, scraped offand suspended into the preparation. Microbes at different growth phasescan be used. For example, microbes at lag phase, early-log phase,mid-log phase, late-log phase, stationary phase, early death phase, ordeath phase can be used.

The formulations comprising the bacterial or fungal endophyticpopulation of the present invention typically contains between about 0.1to 95% by weight, for example, between about 1% and 90%, between about3% and 75%, between about 5% and 60%, or between about 10% and 50% inwet weight of the bacterial or fungal population of the presentinvention. It is preferred that the formulation contains at least about10³ CFU per ml of formulation, for example, at least about 10⁴, at leastabout 10⁵, at least about 10⁶, at least 10⁷ CFU, at least 10⁸ CFU per mlof formulation.

Populations of Seeds

In another aspect, the invention provides for a substantially uniformpopulation of seeds comprising a plurality of seeds comprising thepopulation of endophytes capable of promoting germination, as describedherein above. Substantial uniformity can be determined in many ways. Insome cases, at least 10%, for example, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 90%, at least 95% or more of the seeds in thepopulation, contain the endophytic population in an amount effective tocolonize the plant disposed on the surface of the seeds. In other cases,at least 10%, for example, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 90%, at least 95% or more of the seeds in the population, containat least 1, at least 10, or at least 100 CFU on the seed surface or pergram of seed, for example, at least 200 CFU, at least 300 CFU, at least1,000 CFU, at least 3,000 CFU, at least 10,000 CFU, at least 30,000 CFU,at least 100,000 CFU, at least 300,000 CFU, or at least 1,000,000 CFUper seed or more.

In a particular embodiment, the population of seeds is packaged in a bagor container suitable for commercial sale. Such a bag contains a unitweight or count of the seeds comprising the bacterial or fungalendophytic population as described herein, and further comprises alabel. In one embodiment, the bag or container contains a predeterminednumber of seeds. In an embodiment, the bag or container contains atleast 1,000 seeds, for example, at least 5,000 seeds, at least 10,000seeds, at least 20,000 seeds, at least 30,000 seeds, at least 50,000seeds, at least 70,000 seeds, at least 80,000 seeds, at least 90,000seeds or more. In another embodiment, the bag or container can comprisea discrete weight of seeds, for example, at least 1 lb, at least 2 lbs,at least 5 lbs, at least 10 lbs, at least 30 lbs, at least 50 lbs, atleast 70 lbs or more. The bag or container may comprise a labeldescribing the seeds and/or said bacterial or fungal endophyticpopulation. The label can contain additional information, for example,the information selected from the group consisting of: net weight, lotnumber, geographic origin of the seeds, test date, germination rate,inert matter content, and/or the amount of noxious weeds, if any.Suitable containers or packages include those traditionally used inplant seed commercialization. The invention also contemplates othercontainers with more sophisticated storage capabilities (e.g., withmicrobiologically tight wrappings or with gas- or water-proofcontainments).

In some cases, a sub-population of seeds comprising the bacterial orfungal endophytic population is further selected on the basis ofincreased uniformity, for example, on the basis of uniformity ofmicrobial population. For example, individual seeds of pools collectedfrom individual cobs, individual plants, individual plots (representingplants inoculated on the same day) or individual fields can be testedfor uniformity of microbial density, and only those pools meetingspecifications (e.g., at least 80% of tested seeds have minimum density,as determined by quantitative methods described elsewhere) are combinedto provide the agricultural seed sub-population.

The methods described herein can also comprise a validating step. Thevalidating step can entail, for example, growing some seeds collectedfrom the inoculated plants into mature agricultural plants, and testingthose individual plants for uniformity. Such validating step can beperformed on individual seeds collected from cobs, individual plants,individual plots (representing plants inoculated on the same day) orindividual fields, and tested as described above to identify poolsmeeting the required specifications.

In some embodiments, methods described herein include planting asynthetic combination described herein. Suitable planters include an airseeder and/or fertilizer apparatus used in agricultural operations toapply particulate materials including one or more of the following,seed, fertilizer and/or inoculants, into soil during the plantingoperation. Seeder/fertilizer devices can include a tool bar havingground-engaging openers thereon, behind which is towed a wheeled cartthat includes one or more containment tanks or bins and associatedmetering means to respectively contain and meter therefrom particulatematerials. See, e.g., U.S. Pat. No. 7,555,990.

In certain embodiments, a composition described herein may be in theform of a liquid, a slurry, a solid, or a powder (wettable powder or drypowder). In another embodiment, a composition may be in the form of aseed coating. Compositions in liquid, slurry, or powder (e.g., wettablepowder) form may be suitable for coating seeds. When used to coat seeds,the composition may be applied to the seeds and allowed to dry. Inembodiments wherein the composition is a powder (e.g., a wettablepowder), a liquid, such as water, may need to be added to the powderbefore application to a seed.

In still another embodiment, the methods can include introducing intothe soil an inoculum of one or more of the endophyte populationsdescribed herein. Such methods can include introducing into the soil oneor more of the compositions described herein. The inoculum(s) orcompositions may be introduced into the soil according to methods knownto those skilled in the art. Non-limiting examples include in-furrowintroduction, spraying, coating seeds, foliar introduction, etc. In aparticular embodiment, the introducing step comprises in-furrowintroduction of the inoculum or compositions described herein.

In one embodiment, seeds may be treated with composition(s) describedherein in several ways but preferably via spraying or dripping. Sprayand drip treatment may be conducted by formulating compositionsdescribed herein and spraying or dripping the composition(s) onto aseed(s) via a continuous treating system (which is calibrated to applytreatment at a predefined rate in proportion to the continuous flow ofseed), such as a drum-type of treater. Batch systems, in which apredetermined batch size of seed and composition(s) as described hereinare delivered into a mixer, may also be employed. Systems and apparatifor performing these processes are commercially available from numeroussuppliers, e.g., Bayer CropScience (Gustafson).

In another embodiment, the treatment entails coating seeds. One suchprocess involves coating the inside wall of a round container with thecomposition(s) described herein, adding seeds, then rotating thecontainer to cause the seeds to contact the wall and the composition(s),a process known in the art as “container coating”. Seeds can be coatedby combinations of coating methods. Soaking typically entails usingliquid forms of the compositions described. For example, seeds can besoaked for about 1 minute to about 24 hours (e.g., for at least 1 min,at least 5 min, at least 10 min, at least 20 min, at least 40 min, atleast 80 min, at least 3 hr, at least 6 hr, at least 12 hr, or at least24 hr).

Increased Uniformity in Populations of Plants/Agricultural Fields

A major focus of crop improvement efforts has been to select varietieswith traits that give, in addition to the highest return, the greatesthomogeneity and uniformity. While inbreeding can yield plants withsubstantial genetic identity, heterogeneity with respect to plantheight, flowering time, and time to seed, remain impediments toobtaining a homogeneous field of plants. The inevitable plant-to-plantvariability is caused by a multitude of factors, including unevenenvironmental conditions and management practices. Another possiblesource of variability can, in some cases, be due to the heterogeneity ofthe microbial population inhabiting the plants. By providing bacterialor fungal endophytic populations onto seeds and seedlings, the resultingplants generated by germinating the seeds and seedlings have a moreconsistent microbial composition, and thus are expected to yield a moreuniform population of plants.

Therefore, in another aspect, the invention provides a substantiallyuniform population of plants. The population can include at least 100plants, for example, at least 300 plants, at least 1,000 plants, atleast 3,000 plants, at least 10,000 plants, at least 30,000 plants, atleast 100,000 plants or more. The plants are grown from the seedscomprising the bacterial and/or fungal endophytic population asdescribed herein. The increased uniformity of the plants can be measuredin a number of different ways.

In one embodiment, there is an increased uniformity with respect to themicrobes within the plant population. For example, in one embodiment, asubstantial portion of the population of plants, for example at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 90%, at least95% or more of the seeds or plants in a population, contains a thresholdnumber of the bacterial or fungal endophytic population. The thresholdnumber can be at least 10 CFU, at least 100 CFU, for example at least300 CFU, at least 1,000 CFU, at least 3,000 CFU, at least 10,000 CFU, atleast 30,000 CFU, at least 100,000 CFU or more, in the plant or a partof the plant. Alternatively, in a substantial portion of the populationof plants, for example, in at least 1%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 90%, at least 95% or more of theplants in the population, the bacterial or fungal endophyte populationthat is provided to the seed or seedling represents at least 0.1%, atleast 1%, at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or 100% of the total microbepopulation in the plant/seed.

In one embodiment, there is increased genetic uniformity of asubstantial proportion or all detectable microbes within the taxa,genus, or species of the microbe relative to an uninoculated control.This increased uniformity can be a result of the microbe being ofmonoclonal origin or otherwise deriving from a microbial populationcomprising a more uniform genome sequence and plasmid repertoire thanwould be present in the microbial population a plant that derives itsmicrobial community largely via assimilation of diverse soil symbionts.

In another embodiment, there is an increased uniformity with respect toa physiological parameter of the plants within the population. In somecases, there can be an increased uniformity in the height of the plantswhen compared with a population of reference agricultural plants grownunder the same conditions. For example, there can be a reduction in thestandard deviation in the height of the plants in the population of atleast 5%, for example, at least 10%, at least 15%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60% or more, whencompared with a population of reference agricultural plants grown underthe same conditions. In other cases, there can be a reduction in thestandard deviation in the flowering time of the plants in the populationof at least 5%, for example, at least 10%, at least 15%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60% or more, whencompared with a population of reference agricultural plants grown underthe same conditions.

Decreased Uniformity in Populations of Plants/Agricultural Fields

In certain circumstances, decreased uniformity in a population can bedesirable. For example, plants within a population that are not all atthe same developmental stage may not all be negatively affected by abiotic or an abiotic stress event, and as a result, the population as awhole may show a beneficial trait such as increased yield. As anotherexample, a lack of uniformity may allow for the selection ofplants/seeds with a trait that is not present in the other members ofthe population. Therefore, in another embodiment, there is a decreaseduniformity with respect to a physiological parameter of the plantswithin the population. In some cases, there can be a decreaseduniformity in the height of the plants when compared with a populationof reference agricultural plants grown under the same conditions. Forexample, there can be an increase in the standard deviation in theheight of the plants in the population of at least 5%, for example, atleast 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60% or more, when compared with a population ofreference agricultural plants grown under the same conditions. In othercases, there can be an increase intracellular vesiculoid in the standarddeviation in the flowering time of the plants in the population of atleast 5%, for example, at least 10%, at least 15%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60% or more, whencompared with a population of reference agricultural plants grown underthe same conditions.

Commodity Plant Product

The present invention provides a commodity plant product, as well asmethods for producing a commodity plant product, that is derived from aplant of the present invention. As used herein, a “commodity plantproduct” refers to any composition or product that is comprised ofmaterial derived from a plant, seed, plant cell, or plant part of thepresent invention. Commodity plant products may be sold to consumers andcan be viable or nonviable. Nonviable commodity products include but arenot limited to nonviable seeds and grains; processed seeds, seed parts,and plant parts; dehydrated plant tissue, frozen plant tissue, andprocessed plant tissue; seeds and plant parts processed for animal feedfor terrestrial and/or aquatic animal consumption, oil, meal, flour,flakes, bran, fiber, paper, tea, coffee, silage, crushed of whole grain,and any other food for human or animal consumption; and biomasses andfuel products; and raw material in industry. Industrial uses of oilsderived from the agricultural plants described herein includeingredients for paints, plastics, fibers, detergents, cosmetics,lubricants, and biodiesel fuel. Soybean oil may be split,inter-esterified, sulfurized, epoxidized, polymerized, ethoxylated, orcleaved. Designing and producing soybean oil derivatives with improvedfunctionality and improved oliochemistry is a rapidly growing field. Thetypical mixture of triglycerides is usually split and separated intopure fatty acids, which are then combined with petroleum-derivedalcohols or acids, nitrogen, sulfonates, chlorine, or with fattyalcohols derived from fats and oils to produce the desired type of oilor fat. Commodity plant products also include industrial compounds, suchas a wide variety of resins used in the formulation of adhesives, films,plastics, paints, coatings and foams. The above disclosure generallydescribes the present application. A more complete understanding can beobtained by reference to the following specific examples. These examplesare described solely for the purpose of illustration and are notintended to limit the scope of the disclosure. Changes in form andsubstitution of equivalents are contemplated as circumstances mightsuggest or render expedient. Although specific terms have been employedherein, such terms are intended in a descriptive sense and not forpurposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES

Dormancy and germination depend on several processes and factors. Toensure seedling establishment and success, it is important to controlthe underlying processes or conditions. The role of plant genetics,hormones, and different seed tissues have been relatively well studied.The present examples study the endophyte-plant seed relationship,transitting into a root symbiotic stage towards plant maturation.

Example 1 Taxonomy and Physical Properties of the Endophytes

The endophytes used in the synthetic compositions described herein havebeen deposited as follows: International Depositary Authority ofCanada—IDAC (original strains deposited—IDAC, National MicrobiologyLaboratory, Public Health Agency of Canada, 1015 Arlington Street,Winnipeg, Manitoba, Canada, R3E 3R2; receipts and viability in AppendixA) and Saskatchewan Microbial Collection and Database—SMCD (copies ofstrains deposited) International Depository Authority of Canada—IDAC(original strains deposited) and Saskatchewan Microbial Collection andDatabase—SMCD (copies of strains deposited), (see FIGS. 1-6 and Table1). Strains:

(a) IDAC 081111-06=SMCD 2215;

(b) IDAC 081111-03=SMCD 2210;

(c) IDAC 081111-02=SMCD 2208;

(d) IDAC 081111-01=SMCD 2206;

(e) IDAC 200312-06=SMCD 2204; and

(f) IDAC 200312-05=SMCD 2204F.

SMCD 2215 strain was originally isolated as an endophytic bacterium ofPhyalocephala sensu lato plant endophytic SMCD fungus. Classificationaccording to Labeda et al. [2012]. This phylogenetic study examinesalmost all described species (615 taxa) within the familyStreptomycetaceae based on 16S rDNA gene sequences and illustrates thespecies diversity within this family, which is observed to contain 130statistically supported clades. The present 16S rDNA sequence dataconfirm that Streptomyces sp. strain SMCD 2215 can be assigned to aseparate unknown Glade according to Labeda et al [2012] but separatespecies from Streptomyces lividans. Within a plant, it is capable offorming intercellular hyphae-like filaments and intracellular individualspore-like cells.

SMCD2204 is a filamentous, spore-forming facultative endophyte. Within aplant, it is capable of forming hyphal coils, microvesicles,microarbuscules, hyphal knots and intracellular or Hartig net-likestructures. SMCD2204 is a fungus that is capable of being part of aplant-fungus symbiont that produces altered levels of phytohormones,and/or altered levels of anti-oxidants, as compared to a plant that isnot in symbiosis. This endophyte is also capable of being part of aplant-fungus symbiont that shows decreased aging and/or senescence,and/or increased protection against pathogens, as compared to a plant orplant organ that is not in symbiosis.

SMCD2204F is filamentous, spore-forming facultative endophyte that iscapable of living within another endophyte. It is capable of forminghyphal coils and intracellular or Hartig net-like structures within aplant. This endophyte is also capable of being part of a plant-fungussymbiont that shows decreased aging and/or senescence, as compared to aplant or plant organ that is not in symbiosis.

SMCD2206 is a filamentous, spore-forming facultative endophyte. Within aplant, it is capable of forming hyphal coils, microvesicles,microarbuscules, and hyphal knots. SMCD2204 is a fungus that is capableof being part of a plant-fungus symbiont that produces altered levels ofphytohormones, and/or altered levels of anti-oxidants, as compared to aplant that is not in symbiosis. This endophyte is also capable of beingpart of a plant-fungus symbiont that shows decreased aging and/orsenescence, and/or increased protection against pathogens, as comparedto a plant or plant organ that is not in symbiosis.

SMCD2208 is a spore-forming facultative endophyte.

SMCD2210 is a facultative, spore-forming endophyte that is capable ofliving within another endophyte. Within a plant, it is capable offorming hyphal coils and microvesicles. SMCD2210 is a fungus that iscapable of being part of a plant-fungus symbiont that produces alteredlevels of phytohormones, and/or altered levels of anti-oxidants, ascompared to a plant that is not in symbiosis. This endophyte is alsocapable of being part of a plant-fungus symbiont that shows decreasedaging and/or senescence, and/or increased protection against pathogens,as compared to a plant or plant organ that is not in symbiosis.

SMCD2215 is also a facultative, spore-forming endophyte that is capableof living within another endophyte. It is capable of forming hyphalcoils and intracellular or Hartig net-like structures within a plant.SMCD2215 is a bacterium that is capable of being part of a plant-fungussymbiont that produces altered levels of phytohormones, and/or alteredlevels of anti-oxidants, as compared to a plant that is not insymbiosis. This endophyte is also capable of being part of aplant-fungus symbiont that shows decreased aging and/or senescence,and/or increased protection against pathogens, as compared to a plant orplant organ that is not in symbiosis.

Example 2 Symbiotic Microbe-Plant Association and Level of Compatibility

The level of microbe-plant compatibility was assessed using a slightlymodified method of Abdellatif et al. [2009]. In a bicompartmental agar10 cm plate without nutrients (FIG. 7), the plant's health and theformation of root hairs—the absorbants of water and minerals—werecharacterized in co-culture, with and without microbial partners. InFIG. 7, the left compartment of each split plate shows a culture withthe microbial partner, and the right compartment of each split plateshows a culture without the microbial partner. The experiment wasrepeated twice in three replicates.

As shown in the left compartment of each split plate, healthy planttissue formed even when the plant roots were grown directly on the densemicrobial mats. The biomass of root hairs is enhanced to about twice asmuch compared to the right compartment of each split plate where themicrobial partner is absent (see left compartments).

The plant efficacy to establish symbiotic association is dependent onthe type of endophyte distribution within the root endodermis. Typicalendophytic root colonization is discontinuous and partial with a lowernumber of occupied cells<50% (Table 2) compared to the colonization offungal pathogens which is characterized by a uniform/continual(frequency: 60-80%) colonization of cells (FIG. 8).

An endophyte's performance should not only be assessed by measuringbiomass production, because what underlies the visibly increased yieldis the endophyte's efficiency in colonizing the plant. This can beassessed by characterizing their association with plant cells, tissues,or organs (i.e. seed and radicles) using mathematical Indices which havebeen developed [Abdellatif et al. 2009] and applied in this study (FIG.9 and FIG. 10).

These Indices are based on the following observations: Endophyticsymbionts show different radicle (root)-colonization patterns(regularity or level of deviation in endophyte cell form-Ireg anddirection-Idir when colonizing living cell) compared to deadradicle-cell (which usually remain colonized by true saprophytes).

High Ireg and Idir index values determine mutualistic (beneficial)plant-symbiont relationships. In conclusion, the results show that thesymbiotic microbe-plant association is characterised by a high level ofcompatibility between the two partners, leading to an equilibrated (<50%of colonized cortex cells) and discontinuous root colonisation by themicrobial endophytes measured using mathematical indices [Abdellatif etal. 2009]. This mutualistic partnership is further characterised by thedirect effect of endophytic microbes on plant healthy growth (bacto- andmycodependency) when the plant is challenged to use the microbialpartners as the only source of nutrients or energy for growth. Inaddition, the enhancement of the root hairs biomass by the endophyteswas observed and measured even in roots in distal compartments of splitplates where microbial partners were absent, indicating a possiblesystemic plant growth promoting function of the endophytes.

Example 3 Symbiotic Organs of Endophytes on Wheat

Each taxonomical group of endophytes establishes a unique type ofmycovitalism, consequently forming different symbiotic organs.Characterization of the mycovitalism was done using Abdellatif et al.[2009] methodology, consisting of in vitro seed and microbe co-culturesassessing an early stage of the microbe-plant symbiotic association. Thediversity of microbial symbiotic organs formed by SMCD 2204, 2206, 2210,and 2215 on wheat germinants is shown in FIG. 11.

In summary, the results show differential types of symbiotic organsformed in wheat root by each endophyte likely related to their differentsymbiotic functions. An equilibrated colonization abundance, patchycolonization patterns, increased hypha septation in living root cells,as well as formation of arbuscules, knots, coils and vesicles—putativesymbiotic functional organs—may indicate local specialization within thefungal endophytes to promote plant mycovitality and mycoheterotrophy.Bactovitality is mostly characterized by Streptomyces intercellularcurly filaments.

FIG. 66 shows symbiosomes formed in wheat root. The symbiosome is thenew compartment that is formed in the plant cell when bacteria or fungienter it. Symbiosomes can be classified into two types: I and II. Bothtypes are composed of a perivesiculoid membrane and a partiallyfragmented outer vesiculoid membrane. Type I symbiosomes areadditionally composed of an intercellular microvesiculoid compartmentformed between two plant cell membranes, while type II symbiosomes areadditionally composed of an intracellular vesiculoid compartment. Bothtypes can be seed in the form of vesicles (A and B) and knots (C).

Symbiosis at the seed level resulted in increased wheat germinants after10 days of co-innoculation (FIG. 12 and FIG. 13).

Example 4 Endophytes Improve Wheat Seed Germination Under Heat andDrought Stress

Seed germination is a critical life stage for plant survival and timelyseedling establishment especially in stressful environments. It washypothesized that endophytes would improve wheat seed germination underheat and drought stress. The hydrothermal time (HTT) model ofgermination is a conceptual model useful for predicting the timing andenergy of germination (EG) under a given set of conditions. The HTT andEG are applied to determine if one or more compatible endophytes enhanceheat or drought tolerance in wheat. Endophytes tested dramaticallyincreased the percent of germination, improved EG and HTT values, anddiminished wheat susceptibility to heat and drought as measured by freshweight of seedlings. When colonised by the most effective endophyte, thevalues of the parameters tested in wheat seeds exposed to heat stressresembled those of unstressed seeds.

Materials and Methods Hydrothermal Time Model of Germination and Energyof Germination

The hydrothermal time (HTT) model [Gummerson 1986] postulates that anindividual seed begins to germinate when two conditions are met. First,the sum of daily temperatures, above a minimum cardinal value (T_(min)),accumulated over a period of time, must pass a threshold value (θ_(T)),measured in degree days. Second, the seed must accumulate sufficientwater potential (θ_(H)) per degree-day. Thus, HTT (θ_(HT)) can beexpressed as:

θ_(HT)=(θ_(H))(θ_(T)).  (Equation 1)

According to Köchy and Tielborger [2007],

θ_(T)=(T _(substrate) −T _(min))t  (Equation 2)

with t representing the time elapsed in days, and

θ_(H)=ψ_(substrate)−ψ_(min)  (Equation 3)

in a constant environment assuming that T_(substrate) is equal to orless than the optimal temperature for seed germination. In Equation 3,ψ_(substrate) and ψ_(min) represent the water potential of the substrateand the minimum water potential at which germination is possible, inMPa, respectively. Consistent with Bradford [2002], equations 2 and 3can be substituted into equation 1 to yield:

θ_(HT)=(ψ_(substrate)−ψ_(min))(T _(substrate) −T _(min))t  (Equation 4).

However, in the present study, the temperature exceeds the optimaltemperature for the germination of wheat [reviewed by McMaster (2009)],necessitating the consideration of a maximum temperature (T_(max)) abovewhich germination cannot occur. Thus, equation 2 was modified to:

θ_(T)=√[(T _(substrate) −T _(min))(|T _(substrate) −T_(max)|)]t  (Equation 5)

where T_(min)≦T_(substrate)≦T_(max). If equation 5 is substituted for 2in equation 4, the following results:

θ_(HT)=(ψ_(substrate)−ψ_(min))√[(T _(substrate) −T _(min))(|T_(substrate) −T _(max)|)]t  (Equation 6)

where T_(min)≦T_(substrate)≦T_(max).

Energy of germination (EG) can be defined in several ways, including thepercentage of seeds germinating after a set time period after planting,relative to the number of seeds tested [Ruan et al. 2002; Dong-dong etal. 2009], or 50% of germination attained [Allen 1958]. In order tointegrate EG with the HTT model of germination the latter definition wasused, meaning that EG is equal to t in Equation 2.

Estimation of Parameters

The estimation of T_(min) and T_(max) for wheat was based on bothinformation available in the literature and the present inventors' ownobservations. McMaster [2009] summarizes data originating from Friend etal. [1962], Cao and Moss [1989], and Jame et al. [1998] indicating theexistence of a curvilinear relationship between wheat development rateand temperature. Since germination and development of wheat does nottake place below 0° C. or above 40° C., T_(min) and T_(max) wereassigned the values of 0° C. and 40° C., respectively.

The parameter ψ_(min) was estimated in vitro by germinating wheat seedsgrown on potato dextrose agar (PDA; Difco) media containing a range ofpolyethylene glycol (PEG) 8000 concentrations (Amresco Inc.). The wateractivity (a_(w)) of PDA alone and PDA containing 8%, 12% and 16% PEG wasmeasured using the AquaLab 4TE, Series 4 Quick Start, Decagon Devices.Water activity was converted to water potential (ψ) using therelationship adapted from Bloom and Richard [2002]:

Ψ=[(RT)ln(a _(w))]/V  (Equation 7)

where R is the universal gas constant (8.314 J mol⁻¹ K⁻¹), T is thetemperature in ° K, and V is the partial molar volume of water (18mL/mol). For unit conversions, 1 J/mL=1 MPa=10 bar. Water potential iszero for a free water surface or a saturated medium; all other valuesare negative.

The water activities of PDA and PDA containing 8%, 12% and 16% PEG were0.9974, 0.9890, 0.9863, and 0.9825, respectively. These values areequivalent to −0.35, −1.51, −1.88, and −2.41 MPa, respectively and areconsistent with those reported in the literature [Leone et al. 1994].

Plant and Fungal Material

The plant material used was the durum wheat cultivar AC Avonlea, whichhas low resistance to environmental stressors [SaskSeed guide 2008]. Theseeds used in the first round of experiments were produced by PatersonGrain in 2008, under field conditions, and not certified to be free ofmicrobes. Seeds used in the second set of experiments were produced bythe Agriculture and Agri-Food Canada (AAFC) Seed Increase Unit ResearchFarm in 2006 under greenhouse conditions, and were certified to be freeof microbes. Wheat seeds were surface-sterilized with 95% ethanol for 10s, rinsed in sterile distilled water for 10 s, submerged for either 3min (first round of experiments involving seeds not certified to be freeof microbes) or 1 min (second round of experiments using seeds certifiedto be microbe-free) in 5% sodium hypochlorite (Javex), rinsed threetimes in sterile distilled water and PDA for germination [Abdellatif etal. 2009]. A third seed sterilization method, involving a 3 hr exposureto chlorine gas (produced by combining 25 mL 6% sodium hypochlorite with1.0 mL concentrated hydrochloric acid in a beaker) in a closed plasticbox placed in a fumehood [Rivero et al. 2011] was also tested. Thepercent germination of seeds subjected to each sterilization protocoland placed on PDA for three days is shown in FIG. 14B. Only the 3 minsubmersion in sodium hypochlorite resulted in a significant decrease ingermination (p≦0.01). Seed surface sterilization was intended toeliminate microbes which could compete with the endophytes beinginvestigated. In addition, microbes present on the surface of the seedscould overgrow the plate and emerging seedling, inhibiting plant growth.All seeds used in the study were determined to be free frommicroorganisms after sterilization, based on the absence of unintendedmicrobial growth on the plate.

Four endophytic Ascomycota mitosporic fungal isolates (classifiedaccording to Kiffer and Morelet [2000]): SMCD 2204, SMCD 2206, SMCD2208, and SMCD 2210, plus the Actinomycetes filamentous gram positivebacterial isolate SMCD 2215; compatible with Triticum turgidum L.[Abdellatif et al. 2009] were used in this study. Endophytes were grownon PDA for at least three days at room temperature in darkness prior toexperimental use.

Endophytes as Free-Living Organisms

Agar plugs (5 mm²) cut from the margins of the parent colony were placedin the centre of a 90-mm Petri dish containing either PDA alone oramended with 8% PEG (drought). The Petri dish was sealed with parafilm(Pechiney Plastic Packaging) to maintain sterility and placed in abench-top incubator (Precision Thermo Scientific, model 3522) at either23° C., or under heat stress, 36° C., in darkness. The diameter of thecolony was measured at 24, 48, 72, 96 h, and five and six days. Thechanges in diameter were used to calculate colony growth rate. Thegrowth of a minimum of three replicates per isolate was measured.

Endophytes Ability to Confer Heat and Drought Tolerance to Wheat

Each isolate was applied individually to wheat seeds prior togermination according to the method described in Abdellatif et al.[2010] and shown in FIG. 14A. Briefly, five surface-sterilized seedswere placed at a distance equivalent to 48-h hyphal growth from a 5mm²-agar plug, placed hyphal side down in the centre of a 60-mm Petridish. For slow growing isolates, the agar plug of endophyte colony wasplaced in the Petri dish one to four days prior to the introduction ofthe seeds. The seedlings were germinated for one week under abioticstress and control conditions.

Drought stress was induced using PDA containing 8% PEG. Heat stress wasinduced in a bench-top incubator in darkness; the temperature wasgradually raised by 2° C. every 2 h from 28° C. to 36° C. In the initialround of experiments, percent germination at three days and fresh weightat one week was assessed. Each experiment consisted of six Petri platesand was repeated, independently, three times. In subsequent experiments,percent germination was assessed every 24 hrs for seven days. Eachexperiment consisted of 10 Petri plates and was repeated either twice(heat and drought stress combined) or three times (heat stress, droughtstress and control conditions).

The stable internal colonization of wheat roots by the intendedendophytes was confirmed by re-isolation of the endophytic organism fromroots which had been surface sterilized to remove an external microbialgrowth using a procedure modified from Larran et al. [2002]. Rootfragments (˜0.5 cm) were surface sterilized in 95% ethanol for 10 s,rinsed in sterile distilled water for 10 s, submerged for 20 s in 5%sodium hypochlorite (Javex), rinsed three times in sterile distilledwater and placed on PDA in a 60 mm diameter Petri dish. The Petri dishwas sealed with parafilm and incubated in the dark at room temperaturefor four to seven days prior examination.

Statistical Analysis

The colony growth rates of free-living endophytic organisms grown underheat or drought stress were compared to those of the same organism grownunder control conditions using analysis of variance (ANOVA) followed bypost-hoc Fischer's' least significant difference (LSD) test. Percentgermination data was subjected to arcsine transformation prior tostatistical analysis [McDonald 2009]. Statistical differences betweenpercent germination after both three and seven days, and fresh weight atseven days were assessed using a single factor ANOVA to compare alltreatments. Subsequently, a post-hoc LSD test was used to evaluate thesignificance of differences between the no endophyte control and seedstreated with each mycobiont. The level of statistical significanceassociated with differences between the EG and HTT required to reach 50%germination of endophyte-colonized and control seeds were assessed byevaluating the EG for each of the three independent replicates of theexperiment. The resulting data were subjected to an ANOVA and post-hocLSD analysis. P-values less than 0.05 and 0.01 were considered to besignificant and highly significant, respectively. Statistical tests wererun with SPSS Inc. 2011.

Results

Within each section, the results are organised according to the type ofstress: heat, drought, heat and drought, or no stress. Within eachstress, the results dealing with plant material are presented accordingto the germinant and/or seedling traits measured: percent germination atthree and seven days, fresh weight at seven days, EG and HTT.

Free-Living Endophytes

The phenotypes of SMCD 2206, 2210 and 2215 were not altered by heat (36°C.), while SMCD 2204 and 2208 did not grow at 36° C. The colony growthrates of SMCD 2206 and 2210 were reduced by 36° C. as compared tonon-stressed conditions (p 0.01), while the growth rate of SMCD 2215 at36° C. was increased (p≦0.05) (FIG. 15). At 36° C. SMCD 2215 grew themost rapidly, followed in decreasing order by 2206 and 2210 (FIG. 15).

The morphology of SMCD 2204, 2206, 2208 and 2215 was not appreciablyaltered by drought (8% PEG). However, when SMCD 2210 was exposed todrought, this organism lost its “woolly” appearance and instead acquireda “shiny” or “slimy” appearance. The colony growth rates of SMCD 2204,2206, and 2208 were reduced by drought (p≦0.01, p≦0.01, and p≦0.05respectively), while the rate of colony growth of all other endophytesremained unchanged (FIG. 15). When drought stress was applied, SMCD 2204grew at the highest rate followed in decreasing order by 2206, 2210,2208 and 2215 (FIG. 15).

When challenged by 36° C. heat and drought (8% PEG) simultaneously, SMCD2204, and 2208 failed to grow, while SMCD 2206, 2210 and 2215 grew at asignificantly slower rate than under control conditions (p≦0.01) (FIG.15). In control conditions, SMCD 2204 grew the fastest, followed indecreasing order by SMCD 2206, 2210, 2208 and 2215 (FIG. 15).

Response of Endophyte-Colonized Wheat to Heat

At 36° C., colonization by SMCD 2206 and 2215 increased germinationafter three days (p≦0.05 and p≦0.01, respectively; FIG. 16A), whereasSMCD 2204, 2208 and 2210 did not alter this parameter (p≧0.1; FIG. 16A).After seven days, 63% and 56% of seeds germinated in co-culture withSMCD 2204 and 2208, respectively. These values were not statisticallydifferent (p>0.1) from the 59% germination achieved by the uncolonizedcontrol. In contrast, the endosymbionts SMCD 2206, 2210 and 2215promoted germination after seven days (p≦0.01; FIG. 17).

When subjected to 36° C., the fresh weight of wheat seedlings was stablein co-culture with SMCD 2204, 2206, 2208, and 2210, while SMCD 2215significantly increased this parameter (p≦0.01 respectively; FIG. 16D).

The EG for wheat seeds co-cultured at 36° C. with fungal endophyte SMCD2210 (p≦0.05; Table 3, FIG. 17) improved compared to endophyte-freeseeds. However, SMCD 2204, 2206, 2208 and 2215 did not alter EG (p>0.1;Table 3) relative to the control. SMCD 2210 augmented the EG to thegreatest extent, followed by SMCD 2206 and 2215 (Table 3). SMCD 2210reduced the time required for 50% of seeds to germinate to a mere twodays.

When exposed to heat stress, the HTT required for germination wasreduced for wheat seeds colonized by SMCD 2210 (p≦0.05; Table 3), butnot any of the other endophytes tested (p>0.1; Table 3). Endophyte-freewheat seeds needed 50 MPa ° C. days more than seeds colonized by SMCD2210 (the most effective endophyte tested) to achieve 50% germination(Table 3). There was a clear, negative, linear correlation between theHTT necessary for 50% germination and the percent germination afterseven days under heat stress (FIG. 18).

Response of Endophyte-Colonized Wheat to Drought

When subjected to drought stress for three days, a diminished percentageof wheat seeds germinated in co-culture with SMCD 2208, compared toendophyte-free seeds (p≦0.01; FIG. 16B), while SMCD 2204, 2206, 2210,and 2215 did not alter this trait (p>0.1; FIG. 16B). After seven days,treatment with SMCD 2206, 2210 and 2215 led to an increase in seedgermination (p≦0.01, p≦0.05, and p≦0.01, respectively; FIG. 17). Incontrast, 65 and 67% of seeds co-cultured with SMCD 2204 and 2208 hadgerminated after seven days. Neither of these values differedstatistically from the 59% of uncolonized seeds that germinated underthe same conditions (p>0.1). Under drought conditions, SMCD 2208 and2210 decreased fresh weight after seven days (p≦0.05 and p≦0.01.respectively; FIG. 16E). None of the other mycobionts altered thisparameter (p>0.1; FIG. 16E).

The EG decreased for wheat seeds co-cultured in drought conditions withall endophytes tested, as compared to endophyte-free seeds (0.05≦p≦0.1for SMCD 2204 and 2208 and p≦0.05 for 2206, 2210 and 2215; Table 3).SMCD 2206 improved the EG to the greatest extent, decreasing the timeelapsed before 50% germination was achieved after 2.6 days (Table 3;FIG. 17).

The HTT required for germination was reduced for wheat seeds treatedwith all endophytes tested under drought stress (Table 3). Whileuncolonized seeds needed 80 MPa ° C. days to achieve 50% germination,seeds colonized by endophyte SMCD 2206 (the most effective endophytetested) required only 34 MPa ° C. days, representing a drop of 46 MPa °C. days (Table 3). There was a visible, negative, linear correlationbetween the HTT required for 50% germination and the percent germinationat seven days under drought stress (FIG. 18). However, the R² valueassociated with this linear relationship was smaller than for thecorrelation found under heat stress. The ranges of HTTs needed toachieve 50% germination differ between heat and drought stress, withvalues between 34 and 44 MPa ° C. days and 80 and 94 MPa ° C. days beingunique to seeds exposed to drought and heat stress, respectively (FIG.18; Table 3). The ranges of percent germination after seven days aresimilar between seeds exposed to drought and those subjected to heat,though the germination levels of heat-stressed seeds cover a slightlylarger range (FIG. 18).

Response of Endophyte-Colonized Wheat to Drought and Heat in Combination

Very few wheat seeds germinated when exposed to drought (8% PEG) andheat stress (36° C.) simultaneously (FIG. 17). Colonization byendophytes SMCD 2210 and 2215 increased the percent germination afterseven days (p≦0.01; FIG. 17). On the other hand, SMCD 2204, 2206 and2208 failed to improve this trait (p>0.1). Seeds co-cultured with SMCD2215 (the most beneficial microorganism tested for this parameter)reached 24% germination, four times the level attained by theirendophyte-free counterparts (FIG. 17).

Because neither uncolonized seeds nor those colonized by any of theendophytes reached 50% germination within seven days, EG could not bedetermined and HTT was calculated for 5%, rather than 50%, germination.The time required to reach 5% germination ranged from 24 h to four days.None of the endophytes tested decreased the time required to attain 5%germination or HTT values (p>0.1). Overall, the HTT needed to reach 5%germination varied from 11 to 43 MPa ° C. days (HTT_(mean)=23.9) (FIG.18; Table 3).

The range of HTT values for seeds subjected to both heat and droughtstress were unique, as compared to the HTT values when either heat ordrought was applied alone. There was a negative, linear relationshipbetween HTT required and the percent germination under combined heat anddrought stress. However, the R² value associated with this linearrelationship was smaller than for the correlation found when either heator drought stress was applied individually (FIG. 18).

Response of Endophyte-Colonized Wheat to Control Conditions

Under non-stressed conditions, SMCD 2215 significantly increased seedgermination compared to uncolonized seeds after three days (p≦0.01)(FIG. 16C). SMCD 2206, 2208 and 2210 positively impacted, whereas SMCD2204 did not alter percent of germination. In unstressed conditions,SMCD 2204, 2210 and 2215 increased the fresh weight of wheat seedlingsafter seven days (p≦0.05 and p≦0.01, respectively). Furthermore, SMCD2206 and 2208 showed no impact on the fresh weight as compared touncolonized seedlings (FIG. 16F).

In control conditions, EG and HTT parameters were slightly improved bySMCD 2206 and 2215 endosymbionts (Table 3). Relatively little alterationin EG and HTT parameters was measured associated with non-stressed wheatseeds in co-culture with different isolates.

Example 5 Endophytes Enhance Yield of Wheat and Barley Genotypes UnderSevere Drought Stress

Summary:

Due to climate change and population growth, the development oftechniques increasing agriculture crop tolerance in stressfulenvironments is critical. Inoculation with three symbiotic endophytes,indigenous to the Canadian prairies, increases wheat and barleyresistance to heat or drought stress, as well as grain yield and seedweight. The use of such fungal and bacterial endophytes in the field hasthe potential to increase the seed germination vigour (SGV=differencebetween total percentage of E− germinating seeds and E+germinatingseeds) (FIG. 19, FIGS. 20A and B), and to enhance yield in stress-proneconditions (Table 4; FIGS. 21 A, Ba, and Bb). Evidence supports thatSMCD strains increase seed-vitality and plant vigour (FIG. 22A-D).Overall results demonstrate that the prenatal care of seed usingendophytic microbes, particularly SMCD strains, ensures superior cropyield of wheat and barley genotypes through physiological improvements.

Materials and Methods

Seeds of the wheat and barley cultivars were produced at University ofSaskatchewan experimental plots and Crop Science Field Laboratory(Saskatoon). Visually healthy seeds were surface sterilized in 95%ethanol for 10 s, rinsed in sterile distilled water for 10 s, submergedfor 1 min in 5% sodium hypochlorite (Javex) and then rinsed three timesin sterile distilled water.

The endophytic isolates used in this study were originally isolated fromthe roots of durum wheat Triticum turgidum L. grown at field sites inSaskatchewan, Canada [Vujanovic 2007b]: SMCD 2204, 2206, 2208, 2210,2215. All endophytic isolates are culturable on potato dextrose agar(PDA; Difco) in the absence of a host plant. Isolates were grown on PDAfor three days at room temperature (23° C.) in darkness prior toexperimental use.

The experiment inoculations were done in pots. Each of the endophyticisolates was applied to cereal (wheat and barley) seeds prior togermination according to the method described in Abdellatif et al.[2010]. Briefly, five surface-sterilized seeds were positioned at adistance equivalent to 48 h hyphal growth from a 5 mm² agar plug, placedhyphal side down in the centre of a 2 L plastic pot filled with 300grams (dry weight) of autoclaved, field capacity Sunshine mix 4 pottingsoil. The seeds and agar plug were then covered with a 3.5-4.0 cm layerof Sunshine mix 4. Five seeds were planted per pot and there were twelvepots per treatment. Pots containing plants were placed in a greenhousefor drought stress and control treatments. The pots were arranged in arandomized block design.

Drought stress was induced from May to September when night-day maximumtemperatures in the greenhouse ranged from 18 to 26° C. On sunny days,natural sunlight provided irradiation, while on cloudy or winter dayswith a shorter photoperiod, 1000 watt high pressure sodium light bulbs,suspended from the ceiling roughly 2 m above the plants, supplementedsunlight. In the first experiment, drought stressed and control (wellwatered) plants were grown at 25% soil water content by weight and 100%water retention capacity, respectively. During the experiment controlplants were watered to 100% water retention capacity three times perweek, while drought stressed plants were water to 100% water retentioncapacity weekly. This drought regime was adopted in order to mimic thenatural cycle of drought that can occur during the growing season inNorth American prairies [Chipanshi et al. 2006].

Mature spikes were collected and dry kernels weighed on a Mettler ToledoPG802-S balance in laboratory.

Results and Discussion Increased Wheat Seed Germination Vigour (SGV)

Under in vitro control conditions, SMCD (2204, 2206, 2208, 2210, 2215)treated wheat seeds germinated consistently faster, more uniformly, andwith much higher SGE (seed germination efficacy). The SGV of seedsinoculated with SMCD (E+) was 15% to 40% greater compared to untreated(E−) seeds (FIG. 19), demonstrating SMCD's efficacy in controlling seeddormacy and enhancing seed vigor. Positive effects of SMCD strains onyield of wheat and barley genotypes under severe drought were alsodemonstrated.

Barley genotypes generally show higher drought susceptibility (low DTE(Drought Tolerance Efficacy) values) and lower yield performance thanwheat (Table 4), possibly due to the extreme drought conditions in thegreenhouse more fitting to wheat. In particular, CDC Kendall-two rowbarley, without endophyte (E−), shows high susceptibility to droughtstress compared to other barley genotypes. However, the endophytetreatments (E+) demonstrate a remarkable positive effect on yield of allgenotypes (Table 4). Conferred resistance ranges from low droughtresistant CDC Kendall to highly resistant New Dale genotypes, whereasconferred resistance to wheat was consistently high.

During the maturity stage of wheat and barley, SMCD endophytesdramatically increase the genotypes drought tolerance parameters such asDTE efficacy and yield. SMCD application on Avonlea, the most droughtsusceptible wheat cultivar detected (DTE=16.1), resulted in a highincrease in yield (77%) under drought conditions compared to control orstandard watering. Carberry profited the most from endophytes undercontrol or normal conditions, whereas CDC Utmost VB and BW 423 performedequally well under both dry and control conditions.

In conclusion, combining drought resistant genotypes with compatibleendophytic SMCD 2206, SMCD 2210, and SMCD 2215 microbial symbiontsmaximizes plant drought resistance, an important aspect in ensuring foodsecurity. Without wishing to be bound by theory, this suggests that themost drought susceptible (low DTE values) wheat (FIG. 19A) and barley(FIG. 19B) cultivars will gain the most from the symbiotic associationwhen exposed to the drought stress.

The only exception seemed to be the six row barley genotype Legacyshowing an extremely low DTE=1.1. Although it responded positively tothe endophyte presence with increased yield of 26.9% under controlconditions, it ameliorated yield only for 5% in symbiosis under stress.Thus, this cultivar was excluded from the barley model presented in FIG.19B.

Effect of Individual SMCD Strains on Wheat and Barley Productivity

Individual SMCD strains positively affect the average kernel yield ofeach genotype, although the actual magnitude varies by genotype-straincombination. FIG. 21 presents results obtained under drought conditionsin the greenhouse (FIG. 21: A—Wheat; B_(a)—Barley (two row), andB_(b)—Barley (six row)).

Early seed contact with compatible SMCD isolates is a prerequisite forprotecting crop against drought, resulting in a higher yield orproduction of kernels. SMCD 2206 generally confers the highest degree ofimprovement for most genotypes. However, strain-cultivar specificityensures highest improvements on an individual basis, e.g. Wheat-PT580and Barley-CDC Copeland prefer SMCD 2210; whereas Wheat-BW423 and PT580,as well as CDC Kendall show higher performance and drought resistancewhen inoculated with SMCD 2215.

Results highlight the importance of mycovitalism in stress-challengedwheat and barley seeds, assisting breeders in the making of highlyproductive cultivars capable of withstanding drought conditionssignificantly better than any cultivar alone (FIG. 22: A-D). Upondemonstrated performance of SMCD strains in fields, producers will havegreen symbiotic products to secure crop yield, and the agro-businesswill benefit from a guaranteed level of positive crop outcomesindependent of fluctuations in environmental conditions.

Example 6 Phytotron Heat Stress Experiment on Pulses

This experiment was conducted under phytotron conditions. All seedvarieties were inoculated with endophytes (SMCD 2204F, SMCD 2206, SMCD2210, and SMCD 2215) and without endophytes in pots containing the soilmix. Details about the approaches used for endophyte inoculation onplant are described above under Example 5. Pots containing plants forheat stress were placed in a phytotron Conviron PGR15 growth chamber(Controlled Environments Ltd.) using a randomized block design. Atemperature of about 33° C. was selected for heat stress. Plants wereexposed to this temperature for 8 h, after which time the plants wereexposed to a temperature of 21° C. for 16 h up to 10 days. After heatshock, temperatures were changed to 16° C. for 8 h and 21° C. for 16 h.

Results

In summary, the results show that the efficacy of each tested endophytein conferring heat stress tolerance is related to the particular plantgenotype or host variety (A—chickpea, B—lentil, and C—pea), and that theimprovement in the biomass is associated to a particular plant organ aseach organ: pod (FIG. 23), stem (FIG. 24) and root (FIG. 25), isdifferentially impacted by heat stress.

SMCD 2215 mostly enhanced the biomass of the stem and pod in pea, andthe biomass of root in chickpea. SMCD 2206 increased the biomass of thestem and pod in lentil, and the biomass of root in chickpea, pea, andlentil. SMCD 2210 mostly improved the biomass of the stem and pod inchickpea, and the biomass of root in pea. SMCD 2204F improved thebiomass of pods in most of the tested crops (chickpea, pea, and lentil).The best performer endophyte-crop genotype combination (E+) showed animprovement of about 300% in the biomass of pod, stem, and root comparedto no endophyte (E−) heat stressed control.

Stem:

The following endophytes showed the best response to heat stress:Chickpea: Amit: SMCD 2210. Vanguard: SMCD 2204F; Pea: Golden: SMCD 2215.Handel: SMCD 2215; and Lentil: Glamis: SMCD 2206. Sedley: SMCD 2206.

Pods:

The following endophytes showed the best response to heat stress:Chickpea: Amit: SMCD 2210. Vanguard: SMCD 2204F; Pea: Golden: SMCD2204F. Handel: SMCD 2215; Lentil: Glamis: SMCD 2206. Sedley: SMCD 2204F.

Root:

The following endophytes showed the best response to heat stress:Chickpea: Amit: SMCD 2215. Vanguard: SMCD 2206; SMCD 2215; Pea: Golden:SMCD 2210; SMCD2215. Handel: SMCD 2206; Lentil: Glamis: SMCD 2206;Sedley: SMCD 2204F.

Example 7 Greenhouse Drought Stress Experiment on Pulses

Six seed varieties [Amit, Vanguard (chickpeas), Golden, Handel (peas)and Glamis, Sedley (lentils)] and endophytes SMCD 2204, SMCD 2204F, SMCD2206, SMCD 2210, and SMCD 2215 were used in this study. Theseexperiments were conducted in the greenhouse. After sowing the seed andinoculating endophytes, pots were allowed to stay without water for 14days to mimic severe drought as proposed by Charlton et. al. [2008] andas per the methodology and conditions outlined by Gan et al. [2004].

Results

In summary, the results show that each SMCD strain positively affectsseveral agricultural parameters on pod production or yield (FIG. 27),and biomass of stem (FIG. 26) and root (FIG. 28) in chickpea (A), pea(B), lentil (C) and under drought stress. Overall, crop genotypescolonised by the symbiotic endophyte (E+) became more resistant todrought vs. heat stress. The level of efficacy of the tested endophytesin conferring drought tolerance varied with the particular plant organ:the pod yield was highly improved in Glamis by SMCD 2204, in Vanguard bySMCD 2204F, in Sedley by SMCD 2206, in Golden by SMCD 2210, and inHandel by SMCD 2215.

Stem:

The following endophytes showed the best response to drought stress:Chickpea: Amit: SMCD 2204F, Vanguard: SMCD 2206; Pea: Golden: SMCD 2204,Handel: SMCD 2204; SMCD 2210; SMCD 2215; Lentil: Glamis: SMCD 2204F;SMCD 2206. Sedley: SMCD 2204F; SMCD 2206.

Pods:

The following endophytes showed the best response to drought stress:Chickpea: Amit: SMCD 2204; SMCD 2210. Vanguard: SMCD 2204; SMCD 2206;SMCD 2215; Pea: Golden: SMCD 2210; SMCD2215. Handel: SMCD 2204F; SMCD2206; SMCD 2215; Lentil: Glamis: SMCD 2204F; SMCD 2206. Sedley: SMCD2210; SMCD2215.

Root:

The following endophytes showed the best response to drought stress:Chickpea: Amit: SMCD 2204; SMCD 2215. Vanguard: SMCD 2204F; SMCD 2206;Pea: Golden: SMCD 2204F; SMCD2215. Handel: SMCD 2204F; Lentil: Glamis:SMCD 2204F; SMCD 2206; SMCD 2210. Sedley: SMCD 2206; SMCD 2210.

Example 8 Streptomyces sp. SMCD 2215 Increases Rhizobium Activity andNodulation Frequency in Peas Under Heat Stress

As was recently observed for another Streptomyces species, S. lydicusWYEC10 [Tokala et al. 2002], the Streptomyces sp. nov. SMCD2215colonizes the roots of young pea seedlings from seeds produced fromplants grown under control conditions. It specifically enhances plantflowering and pod yield (FIG. 29), and root nodulation by Rhizobium sp.(FIG. 30), a native endophytic colonizer of pea seeds discovered in thisstudy (Table 5). Vegetative hyphae of Streptomyces sp. nov. SMCD2215colonize the cells of emerging nodules as discovered by culture plate(PDA), fluorescence microscopy (Carl Zeiss Axioskop 2) and PCR (BioRad)amplification methods [Schrey and Tarkka 2008]

Example 9 Endophytes Confer Abiotic Stress Tolerance to Pulses ViaEnhanced Seed Viability

Pulse crops refer to a group of more than sixty different grain legumecrops grown around the world. The seeds of pulse crops are important tohuman nutrition. The chief constraints to pulse production are bioticand abiotic stresses such as drought, heat, cold and salinity. Recentresearch suggests that endophytic microbe-plant interactions are aninstrumental determinant of plant adaptation.

This study hypothesizes that endophytes increase the rapidity anduniformity of seed germination under optimal and stress conditionsin-vitro. The aim was, firstly, to measure the intrinsic symbioticcapacity of endophytes to trigger germination; and, secondly, to measurethe efficiency of the compatible endophytes in conferring heat anddrought resistance to pulses genotypes.

Material and Methods

Two varieties of pulses, Glamis (lentil) and Handel (pea), wereco-cultured with compatible SMCD 2206 and SMCD 2215, fungal andbacterial symbiotic strains, respectively. The endophytic strains'ability to confer stress tolerance to Glamis (FIG. 31) and Handel (FIG.32) genotypes were tested during in-vitro seed germination modellingdrought (6% PEG) and heat (33° C.) environments.

Seeds were surface sterilized with 95% ethanol for 20s, rinsed twice insterile distilled water for 10s followed by 2 min in 3% sodiumhypochlorite (Javex). Finally, seeds were rinsed in sterile distilledwater 4 times. Seeds were inoculated on PDA media with and withoutendophytes in the dark at room temperature [Abdellatif et al. 2009].Microbial organisms were grown on PDA for at least three days at roomtemperature in darkness prior to experimental use. The endophyticability to confer plant stress resistance was assessed using the energyof germination, which is meant to capture the temporal nature ofgermination and which is defined as the number of days required to reach50% of germinating seeds.

Results

The present study demonstrates the differential capacity of fungal orbacterial endophytes to confer drought and heat resistance in pulsesspecific to a fungal or bacterial strain-plant genotype-abiotic stresscombination. This study used molecular and proteomic analyses to betterunderstand the mechanism by which endophytes confer symbiotic stressresistance to pulses.

SMCD strains significantly increased the frequency of pulse seedgermination under standard in-vitro conditions (FIGS. 31 and 32). Understressful conditions, both endophytes (SMCD 2206 and SMCD 2215)increased the frequency of germination when compared to non-colonizedseeds. Frequency of germination was from 70-100% in symbiotic treatmentsand 60-80% germination in the control, meaning that the testedendophytes have the potential to increase seed germination vigour (SGV)by >15%. The highest frequency of germination (100%) was observed inGlamis (lentil) associated with both SMCD 2206 and SMCD 2215 underdrought stress vs. heat stress. When co-inoculated with SMCD strains,the energy of germination (>50% germinating seeds) in Glamis wasachieved in 2 days under drought and in 3 days under heat conditions.Similar results were achieved in Handel (pea), except that this genotypehas inherently a higher ability to support heat shock than Glamis(lentil).

Example 10 Endophytes Enhance Yield of Flax and Canola Genotypes UnderSevere Drought Stress in Greenhouse Experiment

The aim of this study was to use three randomly selected isolates (SMCD2206, SMCD 2210 and SMCD 2215 and to expand the efficiency test on flaxand canola yield production under drought stress.

Material and Methods

The experimental design, flax (Bethun and Sorel) and canola (1768S) seedmanipulation, endophytic inoculant (SMCD 2206, SMCD 2210 and SMCD 2215)application, drought conditions, and yield assessment are as detailedunder Example 5 with small modifications. Briefly, control plants werewatered to 100% water retention capacity three times per week, whiledrought stressed plants were watered to 100% water retention capacityweekly. This drought regime was adopted in order to mimic the naturalcycle of drought that can occur during the Canadian prairie growingseason in which no precipitation falls for seven consecutive days, ormore.

Results and Discussion

Severe drought conditions compromised non-symbiotic flax yield, whileendophytic inoculants SMCD 2206 and SMCD 2210 dramatically improved flaxyield in these same conditions. In particular, under drought conditions,SMCD 2206 maintains a nearly 100% yield in Bethun while SMCD 2210provides a 50% yield compared to the unstressed control in thegreenhouse (FIG. 33). In terms of canola, an improved yield wasregistered in combination with SMCD 2210 (>100%), followed by SMCD 2206(˜50%) and SMCD 2215 (˜30%) compared with unstressed control (FIG. 34).

The bioprotection capacity was also tested in greenhouse againstFusarium avenaceum and F. graminearum. Autoclaved seeds were infected byFusaria inoculants in darkness for 7 days at 25° C. (FIGS. 35-36), andwere inoculated by endophytes produced on petri plates as described byAbdellatif et al. [2009].

Mixed pot soil was inoculated with twenty seeds bearing Fusarium. Thecomposition of mixed soil was 55-65% Canadian Sphagnum Peat Moss,Perlite, and Limestone mixed with sand. Standard greenhouse conditionswere 8 h day light interchanged with a 16 h photoperiod (1000 lux)regime under a relative humidity of 70% and a constant temperature of25° C.±2° C.

Plants treatments were as follows:

T1: Untreated plants (control)

T2: Plant+endophyte

T3: Plant+pathogen, Fusarium avenaceum

T4: Plant+pathogen, Fusarium graminearum

T5: Plant+endophyte fungus+Fusarium avenaceum

T6: Plant+endophyte+Fusarium graminearum

Each treatment was replicated in three pots, and seedlings were wateredthree times a week under controlled conditions. The endophyte-rootcolonisation was tested using a fluorescence microscope to distinguishsymbiotic vs. pathogenic endophyte-wheat relationships [Abdellatif etal. 2009].

FIGS. 37-40 show the positive effect of endophytes on wheatpost-emergency seedling resistance (FIG. 37), foliage and root biomass(FIG. 38 and FIG. 39), and flowering/anthesis stage and spikes (FIG. 38,FIG. 39, and FIG. 40). All tested endophytes induced well-developedfoliage compared to control, as well as well-developed flowers in thepresence of endophytes.

To confirm the ability of the endophytes to stimulate mature plantgrowth in the presence of Fusarium pathogens, the performance of theflowering stage bearing the spikes we assessed as a more advanced growthstage.

The histograms in FIG. 41 illustrate the performance of endophytes inimproving the biomass or dry weight of wheat spikes after doubleinoculation (SMCD endophyte and Fusarium pathogen).

The yield of wheat in the presence of an endophyte and Fusariumsignificantly improves using all endophytic strains compared withtreatment infected with F. graminearum and F. avenaceum but without anendophyte (E−) (FIG. 42). Plants treated with the pathogen alone show asignificantly lower size of spikes compared to control plants and plantswith endophytes (E+) (FIG. 42).

Example 11 Endophyte-Mediated Abiotic Stress Resistance Gene Expressionin Pulses Abstract

The genomic and proteomic mechanisms of plant endophytes beneficialeffects on host plant resistance to abiotic stressors are poorlyunderstood. One of the contemporary theories suggests that the symbioticplants are protected from oxidative stress produced by heat, drought andsalt stressors by the production of antioxidant molecules. The aim ofthis study is to shed more light on defensive symbiosis of pea, chickpeaand lentil genotypes assessing the Pro, SOD, and MnSOD gene expressionstriggered by the association between host genotypes and endophytes. Theresults of this study demonstrated endophyte-mediated gene expression inendophyte-inoculated plants. These genes play an important role andprovide the host protection through an enhanced stress tolerance to thetested abiotic stressors.

Materials and Methods

Leaves were collected for this analysis from normal and stressed 6 seedvarieties (Amit, Vanguard [chick pea] (FIG. 43), Golden, Handel [peas]and Glamis, Sedley [lentils]) with or without endophytes.

Real-Time PCR was used to amplify genes such as Proline (Pro), SOD andMn SOD using primers as shown in SEQ ID NOs: 8-15 (Table 6), stressproteins generally found to play special roles in protecting cytoplasmfrom dehydration and in protecting plants by palliating the toxicityproduced by the high concentrations of ions. PCR was conducted under thefollowing conditions: 3 min at 95° C. (enzyme activation), 40 cycleseach of 30 sec at 95° C. (denaturation) and 30 s at 60° C.(anneal/extend). Finally, a melting curve analysis was performed from65° to 95° C. in increments of 0.5° C., each lasting 5 s, to confirm thepresence of a single product and absence of primer-dimer. Quantitationis relative to the control gene by subtracting the CT of the controlgene from the CT of the gene of interest (ΔCT). The resulting differencein cycle number is then divided by the calibrator normalized targetvalue, and the value obtained (ΔΔCT) is the exponent of base 2 (due tothe doubling function of PCR) to generate the relative expressionlevels.

Results

Different gene expressions during drought stress were analyzed. Table 6shows the genes that were tested. Some of the results obtained fromHandel variety when exposed to 6% PEG.

SOD and MnSOD

In general, SODs play a major role in antioxidant defense mechanisms. Inthe present study very high levels of SOD expression were observed innormal (E−, control) leaves exposed to 6% PEG, an almost 200 foldincrease. Endophytes played a very significant role in decreasing thisstress. Especially, SMCD 2215, followed by and SMCD 2210, SMCD 2204 andSMCD 2206. These symbionts drastically reduced the stress with only a 9and 24 fold increased expression observed (FIG. 44A).

MnSOD is one of the SOD forms. Control leaves showed a 16 fold increasein the gene expression, whereas SMCD 2215 suppressed the stress anddecreased the fold change from 16 fold to 2 fold, followed by SMCD 2206,SMCD 2210 and SMCD 2204 (FIG. 44B).

Proline

Proline is essential for primary metabolism. Proline biosynthesis iscontrolled by the activity of two P5CS genes in plants. This gene wasassessed in Pea variety Handel with endophytes under drought condition.As expected P5CS gene was upregulated and increased expression by 5 foldin the leaves collected from PEG exposed plants. Whereas the leavescollected from seeds associated with SMCD 2206 expressed 2.8 foldfollowed by SMCD 2215 at 3.4 fold expressed proline coding gene (FIG.45). These results confirmed that endophytes play major role in stressresistance modifying proline gene expression compared to uninoculatedstressed plants.

Example 12 Gene Expression Patterns in Wheat Coleorhiza Under Cold andBiological Stratification Abstract

Wheat is one of the widely used major crops in the world. However,global wheat production has decreased about 5.5% in last two decades anda further decline has been predicted due to pervasive global warmingThus, elucidating conditions and techniques that enhance seedgermination is of great importance. Cold stratification is a long-knownmethod of releasing seed dormancy and promoting germination. Biologicalstratification through fungal endophytes can also stimulate seedgermination in many cereal crops. Coleorhiza is one of the most activetissues in seed and it is also the first part to emerge out ofgerminating seeds. To evaluate the efficiency of the stratificationmethods, germination percentage of wheat seeds was assessed under coldand biological stratification and the expression level of gibberellinand abscisic acid genes in coleorhiza were determined Both cold andbiological stratification treatments significantly (P<0.05) enhanced therate and efficacy of germination. Spatial distance between the fungalendophyte and seeds is a determining factor of biological stratificationas seeds in direct contact with fungal endophyte showed highestgermination percentage (up to 86%). High expression of GA3ox2 gene inwheat coleorhiza was found throughout the germination period revealingconsistent production of the bioactive GA3 molecule. The 14-3-3 geneexpression was lowest under endophyte-direct treatment. The expressionof abscisic acid-ABA biosynthesis gene, TaNCED2, was considerably highin cold stratification seeds reflecting the role of abscisic acid as astress-adaptation hormone. High expression of TaABA8′OH1 gene was alsofound in coleorhiza. Overall, this study provides molecular evidence ofthe importance of coleorhiza in germinating wheat seeds. By comparingcold and biological stratification methods, seed germinability can bemarkedly enhanced through application of fungal endophytes, and thespatial distance between seed and endophyte is a factor drivingmycovitality.

Materials and Methods Wheat Seeds

Seeds of the durum wheat cultivar AC Avonlea with low resistance toenvironmental stress conditions were used in this study. These seedswere produced by Agriculture and Agri-Food Canada Seed Increase UnitResearch Farm in 2006 under greenhouse conditions, and were recommendedas free of microbes. Seeds were kept in sterile ziplock bags and storedin 4° C. cold room until further use.

Comparison of Seed Sterilization Protocols

Various methods have been proposed for surface sterilization of wheatseeds. Here four widely acknowledged seed-sterilization methods werecompared to identify the best suitable protocol that efficientlysterilize seed-surface without affecting seed quality and vitality inthis variety of wheat. In the first method, seeds were surfacesterilized with 95% ethanol for 10 s, followed by rinsing in steriledistilled water three times for 1 min [Zhang et al., 2007. BMC Genetics8]. Second protocol was bleach-sterilization where seeds were surfacesterilized in 5% sodium hypochlorite for 3 min followed by thoroughrinsing in sterile distilled water three times for 1 min. In the thirdprotocol, seeds were surface sterilized with 95% ethanol for 10 s,rinsed in sterile distilled water, then submerged for 3 min in 5% sodiumhypochlorite, rinsed three times in sterile distilled water and placedon potato dextrose agar (PDA) for germination [Abdellatif et al. 2009].The fourth method was vapour phase sterilization of seeds with chlorinegas [Desfeux et al., 2000]. In fume hood chamber, a small beaker with 20ml bleach is placed in a 5 litre snaptite box. Wheat seeds were placedin a 96 well-plate and kept in the snaptite box. Then 3 ml ofconcentrated hydrochloric acid was added into the small beaker to createchlorine gas. Lid was kept closed for 4 hours to retain seeds in contactwith chlorine gas. After sterilization, the 96 well-plate was placed for1 hour in a laminar flow hood to disperse trace chlorine gas. Sterilizedseeds were then rinsed three times in sterile distilled water and wereplated out on PDA plates. Comparison of these sterilization methodssuggests that chlorine gas sterilization protocol was the most effectivemethod showing 80% germination without contamination while control seedshad highest percentage of contamination (Table 7). Although bleach andethyl methods successfully inhibited contamination, seed germination wasaffected considerably. Therefore, chlorine gas protocol is a highlyefficient method of sterilization of wheat seeds and it was selected tosterilize the seeds required for experiments conducted in this study.

Cold and Biological Stratification

For cold stratification, surface sterilized seeds were kept on moistfilter paper at 4° C. cold-room for 48 hours [Mukhopadhyay et al., 2004;Wu et al., 2008]. After 2 days, cold stratified seeds were taken to roomtemperature where they were quickly rinsed in sterilized distilled waterand placed on potato dextrose agar (PDA) plates. For biologicalstratification, sterilized seeds were incubated in presence of SMCD2206. Fungal endophyte was grown on PDA at room temperature in darknessfor at least three days before use. To assess this efficiency, wheatseeds were germinated in direct contact and at a certain distance fromthe fungal endophyte. An agar plug (5 mm²) of the endophyte dissectedfrom the margins of a parent colony was placed in the centre of a 90 cmpetri dish with PDA. Then 10 surface sterilized seeds were placed at theperiphery of the petri dish encircling the fungal agar plug atapproximately 4 cm distance. All petri dishes were sealed with 5 layersof Parafilm® (Pechiny Plastic Packaging) to avoid any biologicalcontamination and diffusion of volatile/gaseous compounds. The impact ofdirect-contact of the fungal endophyte was elucidated by placing a 3 mm²agar plug between two adjacent surface sterilized wheat seeds and 5 mm²plug in the centre of the PDA plates. All treatments were carried outwith three replicates of PDA plates with ten surface sterilized seeds oneach plate. Petri dishes were incubated in a bench-top incubator at roomtemperature (−20° C.) in darkness. Incubation time was recorded and datacollection and coleorhiza isolation were carried out after 24, 48, and72-hours.

Germination Percentage

Emergence of early radicles was carefully monitored. Percentage ofgermination was calculated by estimating the number of seeds germinatedout of 10 wheat seeds on each PDA plate. The 50% germination rate wasassumed as the energy of germination. The efficacy of germination indifferent treatments was calculated by following equation:

Efficacy=% germination in a treatment−% germination in control  [Eqn. 1]

Rate of germination was observed for all treated samples and replicates.For Day 2 and Day 3 samples, germination rate was monitored from Day 1to assess the overall vitality. The PDA plates were kept sealedthroughout the data collection period.

Isolation of Coleorhiza

After observing the rate of germination, PDA plates were immediatelytransferred to a sterile biosafety hood chamber for coleorhizaisolation. Wheat seeds were carefully dissected under compoundmicroscope and layers of coleorhiza were cleaved off using sterilizedneedle and scalpel. Isolated coleorhizas were stored in an RNase-freesterilized microcentrifuge tube. Seeds from all biological replicates ofa treatment were combined and approximately 20 to 30 coleorhizas wereisolated to obtain optimum amount plant material for RNA extraction.

RNA Extraction and cDNA Synthesis

To avoid any degradation in plant material, RNA extraction was carriedout forthwith after coleorhiza isolation on each day. Approximately 20mg of coleorhiza samples were taken for RNA extraction. Total RNA wasextracted using Aurum™ Total RNA Mini Kit according to manufacturer'sinstructions (Bio-Rad Laboratories). RNA concentration wasspectrophotometrically measured by Nanodrop (Thermo Scientific)Immediately after RNA extraction, cDNA synthesis was performed usingiScript cDNA Synthesis Kit following manufacturer's instructions(Bio-Rad Laboratories). A 600 ng aliquot of RNA was taken for cDNAsynthesis. Reverse transcription was carried out at 42° C. for 30minutes with a final denaturation at 85° C. for 5 minutes.

Quantitative Real-Time PCR

Expression of gibberellin and abscisic acid functional genes wasestimated by relative quantification using quantitative real-time PCR(QRT-PCR). Various catabolic and biosynthetic genes were selected toassess their respective roles in cold and biological stratification.Wheat actin gene of 131 bp length fragment was used as the internalcontrol [Nakamura et al., 2010]. QRT-PCR was performed using a MJ-MiniGradient Thermal Cycler (Bio-Rad Laboratories) following manufacturer'sinstructions. The PCR condition was 1 cycle of 95° C. for 1 minute and40 cycles of 94° C. for 20 s, 60° C. for 30 s, and 72° C. for 1 min. Forreal-time PCR, cDNA samples from the treatments were used and allreactions were carried out in three replicates and two negativecontrols. Each 25 μl reaction contained 18 μl of iQ™ SYBR® Greensupermix (Bio-Rad Laboratories), 10 pmol of the appropriate forward andreverse primers, 2.5 μl bovine serum albumin, and 25 ng template cDNA.Relative quantification was performed according to Zhang et al. [2007].Expression levels were calculated using cycle threshold (Ct) valuedetermined according to manually adjusted baseline. The differencebetween the Ct values of target gene and actin (Ct^(target)−Ct^(actin))was estimated as Ct and then the expression level was calculated as 2⁻^(Ct) . The mean values of 2⁻ ^(CT) were used to assess difference inexpression between control and stratification treatments. To ensure thespecificity and consistency of amplicons, melting curve analysis andagarose gel electrophoresis were performed after each QRT-PCR run.

Sequencing

Amplicons of Actin and various GA and ABA genes were purified usingBioBasic PCR Purification Kit (Bio Basic Inc.). For each treatment,purified amplicons were sent for sequencing at Plant BiotechnologyInstitute (NRC-PBI). Gene sequences were identified by Basic LocalAlignment Search Tool (BLAST) analyses (http://blast.ncbi.nlm.nih.gov).

Statistical Analysis

One way analysis of variance of germination percentage and geneexpression data was performed using IBM SPSS Statistics software version19. Differences between control and stratification treatments wereexamined with the Duncan's post-hoc test.

Results and Discussion Percentage and Efficacy of Germination

Both cold stratification and biological stratification treatmentssignificantly enhanced the rate of germination with all three treatmentsexhibiting higher germination percentage than control (FIG. 46A; Table8). Endophyte-direct showed highest germination percentage after eachday and increased 60% from Day 1 to Day 3. Throughout the germinationperiod (3 days) it demonstrated significantly (P<0.05) highergerminability than the other three treatments. Only biologicalstratification treatments produced more than 50% germination after Day2. Interestingly, endophyte-indirect treatments showed no germinationafter Day 1 but produced a remarkable 50% germination after Day 2. Coldstratification treatment demonstrated no significant difference fromcontrol after Day 1, and then steadily increased showing significantdifference after Day 2 and Day 3. Pattern of increase in germination isalso reflected in R² values. Whereas control showed an R² value of 0.40,cold stratification and endophyte-direct treatment showed 0.60 and 0.75respectively. On the other hand, owing to its 50% increase from Day 1 toDay 2, endophyte-indirect treatment had the highest R² value of 0.93,which is about 2.5 times higher than control. Energy of germination is acritical parameter determining the capacity of seeds to break dormancyand start germination. Energy of germination is assumed as thepercentage of seed germination after certain time or the number of daysnecessary to achieve 50% germination. Endophyte-direct showed highestefficacy followed by endophyte-indirect and cold stratification (FIG.46B). As there was no germination in endophyte-indirect seeds after Day1, the efficacy of germination was negative. Overall, the stratificationtreatments showed tremendously positive result by reaching 50%germination after 48 hours.

Stratification plays an important ecological role in the release ofprimary dormancy and enhancement of seed germination [Bewley and Black1982; Probert et al., 1989]. Alleviation of seed dormancy andimprovement of germination through cold stratification have beenachieved in many species including grasses [Schutz and Rave 1999],mulberry [Koyuncu 2005], pine [Carpita et al., 1983], tobacco [Wu etal., 2008], rice [Mukhopadhyay et al. 2004], and apple [Bogatek andLewak 1988]. Germination was also increased by cold stratification in 33annual weed species and stratification has been proposed to even becapable of nullifying differences in seed germinability betweenpopulations [Milberg and Andersson 1998]. However, little information isavailable on the impact of cold stratification on wheat seedgermination. This study found that the effect of cold stratificationrequires an initial period and thus seed germination was notsignificantly different from the control on Day 1. However, itdemonstrated considerable impact on germination from Day 2 and thepercentage of germination increased as much as 20% higher than thecontrol. The time period of cold stratification in this study wasselected from previous reports that showed a period of 48 hours iseffective for cold stratification in tobacco [Wu et al., 2008] and rice[Mukhopadhyay et al. 2004]. Earlier studies have shown that the impactof cold stratification is proportional to its time-length [Baskin et al.1992; Cavieres and Arroyo, 2000]. The findings support this and furtherextend the notion to envisage that a slightly longer stratificationperiod (˜4 days) may be required for wheat to attain maximumgerminability.

Several reports have shown the enhancement of seed germination throughthe application of fungal endophytes [Vujanovic 2007b; Hubbard et al.2012; Vujanovic and Vujanovic 2007]. The present study supports theconcept of “mycovitalism”, which is the increase of vitality throughfungal colonization. Fungal endophytes are well known to producevolatile compounds that affect plant phenophases [Mitchell et al., 2009;Strobel et al., 2004]. Thus, endophytes may be capable of affecting seedgermination even when they are not in direct contact with seeds, andthis attribute is particularly useful in field conditions. Here it wasalso tested how physical distance may influence seed germination underbiological stratification. These findings suggest that seeds in directcontact with fungal endophyte are undoubtedly more benefitted than theircounterparts. Endophyte-direct produced highest percentage and efficacyof seed germination on each day of the germination period. Similar toendophyte-direct contact, seeds placed at 4 cm from the endophyte alsogerminated at a significantly higher rate than control. However, thegermination percentage and efficacy were indeed affected by the distanceand indirect-contact seeds have between 14% and 27% less germinationthan direct-contact ones. Furthermore, no germination activity wasobserved on Day 1 which was followed by a sharp rise (50%) on Day 2.Seed germination is an extremely complex process and its underlyingmechanisms are relatively less understood [Nonogaki et al., 2010]. Thusit is not clear how fungal endophytes facilitate the release of dormancyand onset seed germination. Considering fungi are capable of producing arange of plant-growth promoting substances, it is possible thesesubstances are more effective in close vicinity. Consequently,endophyte-direct seeds have significantly higher germination rate thanother treatments. On the contrary, endophyte-indirect seeds showed highefficacy of germination after 48 hours, this period may have allowedenough accumulation of growth promoting substances. There is adifference in germination percentage (6.6%) between the control andendophyte-indirect treatments on Day 1, however, it is not substantial.

Expression Level of Gibberellin and Abscisic Acid Genes in Coleorhiza

The GA3-oxidase 2 and 14-3-3 genes were selected as GA biosynthetic geneand negative regulator of the GA biosynthesis pathway respectively [Jiet al., 2011; Zhang et al., 2007]. The NCED gene is well known for itsrole in ABA biosynthesis pathway whereas ABA 8′-hydroxylase gene isinvolved in ABA catabolic pathway [Ji et al., 2011]. Real-timequantitative PCR analysis indicated that the differential (FIG. 47) andratio expression (FIG. 48) values of distinct functional genes variedsignificantly (P<0.05) among the treatments. Except for the 14-3-3 geneon Day 3, detectable expression was observed for all four genes on eachday. On Day 1, all genes were down-regulated in comparison with control.Expression of GA biosynthesis gene, TaGA3ox2, was considerably higher incold stratification treatment than that of biological stratification. Onthe other hand, 14-3-3 expression did not vary significantly among coldand endophyte treatments although the expression of cold stratificationwas slightly higher than endophytic ones. The transcript level of ABAbiosynthesis gene, TaNCED2, did not vary between control and coldstratification, and was significantly up-regulated than endophytictreatments. The ABA 8′-hydroxylase gene, TaABA8′OH1, showed significantdown-regulation in all three stratification treatments, with lowestexpression observed under cold stratification. The expression patterndid not vary between endophyte-indirect and endophyte-direct treatments.On Day 2, TaGA3ox2 expression was significantly down-regulated in allstratification treatments than control. Expression did not vary betweencold stratification and endophyte-indirect treatments, and lowestexpression was detected in endophyte-direct coleorhizas. No significantdifference was observed for 14-3-3 transcript level among all fourtreatments, although expression was somewhat higher under coldstratification. The expression of TaNCED2 gene was significantly lowerin endophytic treatments than control and cold stratification.Similarly, TaABA8′OH1 gene demonstrated considerable down-regulation instratification treatments than control. The lowest expression wasdetected in endophyte-indirect treatment. The transcript level ofTaGA3ox2 gene also varied significantly among the treatments on Day 3.Cold stratification showed about ten times higher expression thancontrol while two endophytic treatments did not vary significantly.Conversely, TaNCED2 and TaABA8′OH1 genes were significantlydown-regulated in all stratification treatments with lowest expressionin endophyte-direct and endophyte-indirect treatments respectively. Nodetectable expression was observed for the 14-3-3 gene on Day 3.

The ratio of GA and ABA biosynthesis gene expression, TaGA3ox2:TaNCED2,shows no considerable difference among the treatments on Day 1 butsteadily increased thereafter (FIG. 48). Endophyte-indirect exhibitedhighest value on Day 2, which is about 5-10 times higher than the othertreatments; however, all three stratification treatments demonstratedsimilar values on Day 3. Conversely, for the ratio of GA biosynthesisand catabolic genes (TaGA3ox2:14-3-3), endophyte-direct showed highestvalue on Day 1 followed by endophyte-indirect, cold stratification, andcontrol, which is fairly similar to their germination percentage. Theratio of GA biosynthesis and ABA catabolic genes, TaGA3ox2:TaABA1,exhibited similar patterns for all treatments on Day 1, however,endophyte-indirect was considerably higher than others on Day 2. On Day3, cold stratification and endophyte-indirect demonstrated similarexpression level and control was negligible. The ratio between ABAbiosynthesis and catabolic genes (TaNCED2:TaABA1) did not vary among thetreatments throughout the germination period although coldstratification showed slightly higher expression level on Day 1.

Genes encoding GA and ABA biosynthesis and catabolism enzymes showdifferential expression patterns depending on the accumulation oftranscript [Hedden and Phillips, 2000]. Expression patterns of GA3ox1genes have been studied in plethora of plant species includingArabidopsis [Phillips et al., 1995], rice [Oikawa et al., 2004], andwheat [Zhang et al., 2007]. Whereas other GA biosynthesis genes such asGA-20ox are associated with growing vegetative tissues, and flowers,GA3ox (GA3ox2 or GA4H) is exclusively expressed in during seedgermination and supposedly plays a crucial role [Phillips et al., 1995;Yamaguchi et al., 1998; Hedden and Phillips, 2000]. Similar to previousreports, this study also demonstrated high expression of GA3ox2 gene inwheat coleorhiza throughout the germination period. Potentially, withoutwishing to be bound by theory, this reflects consistent production ofthe bioactive GA molecule GA3 in wheat coleorhiza during germination. Onthe other hand, the low expression of 14-3-3 gene, a negative regulatorof GA biosynthesis, was also detected in coleorhiza. With gradualseedling growth and increase in endogenous GA content, the transcriptlevel of 14-3-3 also declined and finally diminished after Day 2.Interestingly, control had highest 14-3-3 level followed by coldstratification, endophyte-indirect, and endophyte-direct, which wassomewhat reflected in their germinability. These results were inaccordance with previous report by Zhang et al. [2007] who showed GAbiosynthesis and catabolic genes closely linked to GA content and shootgrowth.

Expression patterns of the ABA pathway genes have been studied in a widerange of cereals and pulses including rice [Oliver et al., 2007], wheat[Ji et al., 2011; Nakamura et al., 2010], bean [Qin and Zeevart, 1999].The present results show that except control and cold stratification onDay 1, expression of TaNCED2 gene did not vary among treatments.Abscisic acid plays a pivotal role in plant stress-adaptation pathways[Nakamura et al., 2010]. Since the cold stratification seeds were keptat 4° C. for 48 hours prior to their incubation at room temperature, theabscisic acid content may have been higher. On the other hand, highTaNCED2 expression in control may have resulted in higher ABA synthesisand thereby in slower germination rate. Recent reports suggest that thecatabolism of ABA mainly occurs in coleorhiza [Millar et al., 2006;Okamoto et al., 2006]. Furthermore, Barrero et al. [2009] reportedup-regulation and highest expression of ABA8′OH-1 in barley coleorhiza.Similar to these reports, here high expression pattern of TaABA8′OH1gene was found in wheat coleorhiza. The ratio of GA and ABA biosynthesisgenes was fairly linked to percentage of germination. Although,TaGA3ox2:TaNCED2 did not vary remarkably on Day 1, it was highest inendophyte-indirect on Day 2 owing to its significant increase. On theother hand, all three stratification treatments showed considerableup-regulation of TaGA3ox2:TaNCED2 on Day 3, which may have reflected intheir germination.

The underlying mechanisms of biological stratification are stillrelatively unknown but they could reveal how plant-fungus orplant-bacterial interactions take place in the early stages ofgermination. The role of fungal endophytes as bioenhancers is widelyacknowledged [Arnold et al., 2001; Hubbard et al. 2011; Saikkonen etal., 1998; Khan et al. 2012]. In this study, we demonstrated that fungalendophytes can stimulate seed germination significantly, and thismycovitality is proportional to the physical distance between the seedand fungal endophyte. Moreover, the effect of biological stratificationmediated by fungal endophyte is considerably higher than coldpre-treatment. Previous studies have shown that initiation ofgermination is proportional to the time of cold stratification [Cavieresand Arroyo, 2000b] considering this, future study may extend coldstratification period (>48 hours) to increase seed germinability inwheat. Although, cold stratification increased the transcript level ofABA biosynthesis gene, fungal endophytes did not directly stimulate theexpression of phytohormone genes in coleorhiza. However, this studyspecifically assessed the expression of four genes in coleorhiza.

No study has compared germination patterns under cold and biologicalstratification, and elucidated GA and ABA biosynthesis and catabolicgene expression in wheat coleorhiza. Coleorhiza has recently been shownas a highly active component of germinating seed [Barrero et al., 2009].In accordance with this Example, high expression of various functionalgenes in coleorhiza of germinating wheat seeds was also demonstrated.Seed germinability can be substantially enhanced through the applicationof fungal endophytes: 1) via indirect mycovitality or without theendophyte-seed contact on tested distance (for example, the 4 cmdistance was used in this Example) and 2) via direct mycovitality oronce the endophyte reaches seed.

Example 13 Endophytic Stratification Effects on Hormonal Regulators (RSGand KAO) and Resistance MYBs Genes

Stratification is the exposure of seeds to cold and moist conditions inorder to break dormancy, or enhance seed germination. As stratificationis presently limited to the role of abiotic factors, this study aims torender the definition more inclusive by recognizing the role of bioticfactors using mycovitality or bactovitality, or a seed-fungus orseed-bacteria symbiosis as a model. This acknowledges the existence ofboth cold and biological stratifications. Germination of wheat seedssubjected to cold stratification at 4° C. was compared to that ofinoculated wheat seeds at room temperature. Seeds were inoculated withendophytic SMCD2206 strain. Changes in the seed's expression pattern ofplant growth promoting genes—regulators (RSG and KAO) and phytohormonalgibberellins (GAs); and acquired resistance genes (MYBs) in abiotic vs.biotic conditions, during the early breakage of seed dormancy andgermination, were assessed. Measurements were made in the coleorhizacells using qRT-PCR (as described under Example 12). The resultsindicate that the RSG and KAO genes (FIG. 49), coding for enzymespromoting biosynthesis of GAs, and the MYBs resistance genes (FIG. 49)are up-regulated in inoculated seeds. Mycovitality, thus, demonstrates areprogramming effect in pre- and post-germination events of wheat seedtowards enhanced dormancy breakage and germination, effectivelycontributing to the prenatal care of cereal crops.

Material and Methods

RNA samples

This study is the continuation of Example 12. The same material (wheatand SMCD 2206) and in vitro methods as well as the extracted RNA sampleswere used to assess phytohormone RSG and KAO regulators and resistanceMYB gene expresssion by qRT-PCR.

Before RNA extraction started, tubes carried with coleorhiza tissueswere stored in liquid nitrogen immediately as soon as coleorhiza tissueswere isolated to preserve the cells and prevent denaturation of RNA.Aurum™ Total RNA Mini Kit (Bio-Rad Laboratories) was used in total RNAextraction from plant tissues, and it suggested a minimum 20 mg of planttissues were suitable for each sample. The extraction steps were donerapidly and the entire process was kept either in ice, as RNA, wereeasily denatured at room temperature. Fresh extracted total RNA, weredirectly loaded with premixed cDNA synthesis agents obtained fromiScript cDNA Synthesis Kit (Bio-Rad Laboratories). Reverse transcriptionwas carried out at 42° C. for 30 minutes with a final denaturation at85° C. for 5 minutes in a Thermo cycler. cDNA concentration was measuredby Nanodrop spectroscopy (Thermo Scientific) and diluted or concentratedto 100 ng/μl.

Quantitative RT-PCR and Statistical Analysis

The quantitative real-time PCR (QRT-PCR) was performed on a MiniOpticon™Real-Time PCR Detection System (Bio-Rad Laboratories) with iQ™ SYBR®Green supermix kit (Bio-Rad Laboratories). In order to normalize QRT-PCRdata, actin gene (131 bp length fragment) was selected as a referencegene and served as internal control to avoid fluctuation bias of geneexpression under low cDNA concentration [Zhang et al. 2007; Nicot 2005].KAO and RSG gene's primer according to Zhang et al. [2007] were testedin this experiment, whereas original primers were designed for MYB1 andMYB2 based on Triticum aestivum sequences publicly available(http://compbio.dfci.harvard.edu/cgi-bin/tgi/geneprod_(—)search.pl) inComputational Biology and Functional Genomics Laboratory (HarvardUniversity). The MBY newly designed primers (Table 9):

Transcription factor Myb2 mRNA (158 bp) which comprises the sequences asshown in SEQ ID NO:16 and SEQ ID NO:17 and transcription factor Myb1mRNA (152 bp) which comprises the sequences as shown in SEQ ID NO:18 andSEQ ID NO: 19 (Table 9).

100 ng/μl cDNA samples were further diluted to 10 ng/μl and 2 μl cDNAwere used for each 25 μl reaction. In addition, 12.5 μl of iQ™ SYBR®Green supermix, 8.5 μl sterile milli-Q water, 1 μl of each forward andreverse primer (10 pmol) were made up to 25 μl reaction mix. Theprotocol of thermo-cycle was suggested as 95° C. for 10 minutes and 40cycles of 94° C. for 20 s, 60° C. for 30 s, and 72° C. for 1 min. Allthe cDNA samples from the treatments were carried out in threereplicates and two negative controls in QRT-PCR. The gene expressionlevels referred to quantitative curves were carried out by CFX Manager™Software (Bio-Rad Laboratories). Cycle quantification (Cq) value fromthe recorded fluorescence measurements were adjusted manually withbaseline. Relative quantitation is the statistical method chosen in thisstudy [Gizinger 2002]. Gene of interest relative to the endogenouscontrol gene was used to compare with different treatments. Thequantification (ΔCT) was done relative to the subtraction from Cq valueof the gene of interest to Cq value of the control gene. ΔCT was furthersubtracted by calibrator value and generated corresponding ΔΔCT valueswhich were transformed to log 2 (doubling function of PCR) to synthesizerelative gene expression levels [Jurado et al., 2010]. Amplified, RSG,KAO and MYB genes were purified by using BioBasic PCR Purification Kit(Bio Basic Inc.) and sent for sequence job at Plant BiotechnologyInstitute (NRC-PBI). Gene sequences were identified by Basic LocalAlignment Search Tool (BLAST) analyses (http://blast.ncbi.nlm.nih.gov).High identity or similar genes corresponding to different homologousorganisms were assembled and aligned by software MEGAS (MolecularEvolutionary Genetics Analysis). A phylogeny tree was made with thestatistical method of Neighbor-joining based on the aligned genes.

Example 14 Nitric Oxide (NO) Showed the Regulatory Effect onMycovitalism During Early Seed Germination Events

Nitric oxide (NO) is a highly reactive signal molecule common to fungal,animal and plant systems. NO is also known as a signaling moleculeinvolved in eukaryotic cell hormonal signaling [Guo et al. 2003] andplant response to abiotic and biotic stresses [Hayat et al. 2010]. Whilethere is evidence for NO accumulation, increased activation of SOD andproline contributing to the delay of O²⁻ and H₂O₂ accumulation in wheatleaves under salt stress, almost no information exists for fungalendophytes and there interaction with seed germination (mycovitalism).Here, the occurrence of NO in the early stages of germinating wheat ACAvonlea seeds was investigated for three days—endophyte SMCD 2206 onPDA, focusing on the radicle response to fungal diffusible molecules. NOwas visualized in radicle (early root organ) in culture germinants byfluorescence microscopy using the specific probe 4,5-diaminofluoresce indiacetate; the assessment was conducted after five-minute of expositionto the fungal exudate, as sufficient to induce significant NOaccumulation [Calcagno et al. 2012]. Since, SMCD 2206 exudate induced asignificant production of NO in the wheat's root tissues; withoutwishing to be bound by theory, it is possible that this production isregulated by a molecular dialogue occurring in the wheat symbiosis.

Material and Methods

The accumulation of NO in radicle tissues was analyzed in wheat ACAvonlea germinating seed (in vitro approach presented under Example 12)using the cell permeable NO-specific probe DAF-2DA according to Calcagnoet al. [2012] which is converted into its fluorescent triazole derivateDAF-2T upon reaction with NO. The formation of DAF-2T was visualized byfluorescence (Carl Zeiss Axioscop 2) microscopy. AC Avonlea germinantwas assessed at 5 min after treatment with the fungal SMCD 2206 exudatefollowing procedure proposed by Nakatsubo et al. [1998]

The specificity of this response to endophytic SMCD 2206 was confirmedby the lack of response in the non-inoculated radical cells. Theanalyses were repeated in three independent biological replicates.

Results and Discussion

Seed treatment with the fungal exudate can mimic—to some extent—theapproach of endophytic hyphae during the presymbiotic phase of theinteraction, as suggested for AM mycorrhiza in co-culture withArabidopsis roots [Calcagno et al. 2010]. The fungal exudate could,therefore, be confidently used to test whether diffusible fungal signalselicit NO accumulation in the host wheat tissues (FIG. 51) during theearly germination events enhancing mycovitality.

Cellular evidence, therefore, suggests that NO accumulation is a novelcomponent in the signaling pathway that leads to mycosymbiosis relatedwith mycovitalism of wheat seed (FIG. 51). This finding has boththeoretical and practical values in attempts to improve plantprenatal-care using endophytic symbionts.

Example 15 Study of the Effects of Endophytes on Phytoremediation andPhytoreclamation

Phytoremediation is a promising environmental technique. It has beenshown to be cost-effective for reclamation of hydrocarbon/petroleum,salt, heavy metal and radioisotope-contaminated soils. In this study,the effects of coniferous (Picea or Pinus) and deciduous (Salix orPopulus) trees, shrubs (Caragana or Krascheninnikovia), and grasses(Festuca or Elymus) infected (E+) and non-infected (E−) by endophyticorganisms (via plant propagation material, seed or root infection andcolonization) (SMCD 2204, 2206, 2208, 2210 and 2215) on thedecomposition, transformation or degradation of petroleum hydrocarbonsin petroleum contaminated soil will be investigated. Plants will begrown in pots containing petroleum contaminated and non-contaminatedsoils. Plants will be inoculated and incubated for 6 months using thegreenhouse method suggested by Soleimani et al. (2010). Unplanted potswill be used as control. At the end of the experiment, plant-rootcolonization (Abdellatif et al. 2009), soil hydrophobicity (Chau 2012),total petroleum hydrocarbons (TPHs), and polycyclic aromatichydrocarbons (PAHs) contents will be analysed (Germida et al. 2010). Thedifference in E+vs. E− plants root and shoot biomass and leafphotosynthesis will be compared (Hubbard et al. 2012) with PAH and TPHremoval in the rhizosphere of the plants. Unplanted pots will be used ascontrol to calculate the efficacy of symbiotic (E+) plants ondegradation of petroleum hydrocarbons (Soleimani et al. 2010). Theinfected plants will decompose, transform or degrade hydrocarbons andsalts, uptake and accumulate and clean up or eliminate the heavy metalsand radioisotopes in the contaminated site, soil or environment.

Example 16 Seed Coating and Preparation for Field Trials

Fungal strains SMCD 2204, 2204F, 2206, 2208, and 2210, as well asbacterial strain SMCD 2215, were plated from a cryopreserved aliquotonto PDA plates and incubated in the dark at room temperature for 5-10days.

From there, approximately 10 agar plugs of 1 cm² area were inoculatedinto 1 liter of the medium in a 2.8 liter Fernbach flask, and cultivatedfor 10 days at room temperature and 130 rpm. Media were as follows:molasses broth (30 g/l molasses and 5 g/l brewer's yeast) for SMCD 2204,2206, 2208, and 2210; YEP broth (10 g/l peptone, 10 g/l yeast extract,and 5 g/l sodium chloride) for SMCD 2215, and PD broth (30 g/l dextroseand 4 g/l infusion from potato solids) for SMCD 2204F. Typical finalbiomass titers for 10 days of growth were 6-12 g-dry cell weight perliter of culture broth (g-DCW/l).

Fungal biomass containing conidia, chlamydospores, and mycelialfragments was filtered through filter paper using a Buchner funnel undervacuum pressure and washed with sterile water. Biomass was then driedover night at room temperature in a biosafety hood, and ground in aWiley mill through a 425 μm (40 mesh) screen.

For SMCD 2204, 2204F, 2206, 2208, and 2210, wheat seeds were coated withsodium alginate (2%) (1 g of wheat with seeds 37.5 uL of sodiumalginate) followed by coating with a mixture of fungal ground biomasswith talc (1:12 ratio). For SMCD 2215, sonicated liquid culture wasmixed with 2% sodium alginate in a 1:1 ratio followed by coating withtalc.

Example 17 Field-Trial Preparation and Planting

The effects of the previously described microbial hosts (SMCD 2204,2204F, 2206, 2208, 2210, and 2215) were analyzed on 9 different crops:corn, spring wheat, soybean, durum, barley, canola, pea, chickpea, andlentil. Field trials were conducted at the following locations: 1) threesites at University of Saskatchewan (Saskatoon, Stewart Valley, andVanguard, Saskatchewan, Canada) for the following crops: spring wheat,durum wheat, barley, canola, pea, chickpea, and lentil; 2) one site inBrookings, S. Dak., USA for spring wheat and corn; and 3) one site inYork, Nebr., USA for corn.

The size of the test plots were 10 m by 1.5 m at Stewart Walley andVangard, and 2.5 m×2.5 m at Saskatoon (University of Saskatchewan),Saskatchewan Canada, and 5 feet by 50 feet (for wheat), 5 feet by 40feet (for corn) at Brookings, S. Dak., and 5 feet by 40 feet for corn atYork, Nebr., USA.

The varieties shown in Table 10 were used across these test sites.Anhydrous urea was used as fertilizer at Brookings, S. Dak. and York,Nebr. No fertilizer was applied in Saskatchewan, Canada.

Water was applied by using center pivot irrigation three times over thecultivation period at Brookings, S. Dak., and line irrigation was usedin York, Nebr. In Saskatoon, Saskatchewan, Canada, water was providedonce per week with 50% of field capacity until spike formation.Vanguard, Saskatchewan, Canada was a naturally dryland site (brown soil,semi-arid), while Stewart Valley, Saskatchewan, Canada was a naturallymoist site (dark brown soil).

The targeted seeding density, planting date, and harvesting date foreach crop and location are listed in Table 11. Plants were harvestedusing a 5 foot research combine.

Example 18 Quantification of Traits in Field Trials

Flowering time was assessed by visually scoring the plots on the date ofthe first flower opening (bud burst). Data presented shows the change inflowering time (in days) relative to the abiotic formulation control.FIG. 52 shows the early flowering of canola plants treated with themicrobial formulations, as compared to the abiotic formulation control.

Damage resulting from pests (grasshopper, in this case) was assessed byvisually scoring the loss of crop flowers (for canola). FIG. 53 showsthe reduced canola crop damage in plants treated with the microbialformulations, as compared to the abiotic formulation control.

Fusarium head blight (FHB) is caused largely by the Fusarium graminearumspecies in North America. Infection was assessed visually, wheresymptoms of disease in wheat include tan or brown colored lesions thatmay include single spikelets or large sections of the head. FIG. 54shows the reduced incidence of FHB for three spring wheat and one durumwheat varieties, across two sites.

Lower leaf spot and flag leap spot diseases are largely caused byPyrenophora tritici-repentis (tan spot), Stagonospora nodorum(Stagonospora blotch), Septoria tritici (Septoria blotch). This wasassessed visually for wheat and scored on a scale of 1-10 (low to highinfection). FIG. 55 shows the reduced spot disease rating of threespring wheat varieties and one durum wheat variety when inoculated withthe described microbial compositions. These have greener leaves ascompared to the abiotic formulation control, indication protecting fromleaf spot diseases, delayed senescence, or both.

Grain yield (for wheat and corn), in dry bushels per acre, wascalculated by using the weight harvested per plot, the test weight, thepercent moisture, and shrinkage factor. Pod weight was measured forcanola, and total biomass was measured for lentil, chickpea, and pea.FIG. 56 and FIG. 57 show the percentage change in yield (represented bygrain or seed weight, pod weight, or total biomass) of the crops treatedwith the described microbial compositions relative to the abioticformulation control in their corresponding field condition. FIG. 58shows the percentage increase in corn ear weight of the crop treatedwith the described microbial compositions relative to the abioticformulation control in their corresponding field condition.

Example 19 Greenhouse Trials with Additional Crops

A variety of other corn crops were grown with the applied microbialcompositions to assess their ability to affect a variety of planttraits. Tomato, alfalfa, corn, sweet corn, organic corn, swiss chard,radish, and cabbage were all grown in the greenhouse under normal waterand/or drought conditions. For normal water conditions, the plantsreceived water three times per week over the course of two months. Fordrought conditions, the plants received watering three times per weekfor two weeks and no water for the next two months. FIG. 59 shows theeffect of SMCD 2204F, 2206, and 2215 on tomato in shoot length andweight, root length and weight, total plant biomass, and fruit weight.FIG. 60 shows the effect of SMCD 2204, 2206, and 2215 on alfalfa inshoot length and weight, root length and weight, and total plantbiomass. FIG. 61 shows the effects of the described microbialcompositions in the form of total plant biomass under normal water anddrought conditions for corn, sweet corn, organic corn, swiss chard,radish, and cabbage.

Example 20 Mycovitalism Modulates Phytohormone Production in Wheat SeedTreatment and Plant Growth

Phytohormone quantification was undertaken on wheat (CDC Avonlea) plantsin a greenhouse scenario, treated with SMCD 2206 and an untreatedcontrol group. Germination and plant growth was undertaken at 21 C. Thefirst leaves were collected at 7 days post-germination. Green leafsamples were treated with liquid nitrogen, freeze dried, and stored at−80 C until analysis described below.

Tables 12 through 14 describe the phytohormones analyzed in this study.Analysis was performed on a UPLC-ESI-MS/MS utilizing a Waters ACQUITYUPLC system, equipped with a binary solvent delivery manager and asample manager coupled to a Waters Micromass Quattro Premier XEquadrupole tandem mass spectrometer via a Z-spray interface. MassLynx™and QuanLynx™ (Micromass, Manchester, UK) were used for data acquisitionand data analysis.

Extraction and Purification

An aliquot (100 ul) containing all the internal standards, each aconcentration of 0.2 ng/ul, was added to homogenized sample(approximately 50 mg). 3 ml isopropanol:water:glacial acetic acid(80:19:1, v/v/v) were further added, and the samples were agitated inthe dark for 24 hr at 4 C. Samples were then centrifuged and thesupernatant was isolated and dried on a Buchi Syncore Polyvap (Buchi,Switzerland). Further, they were reconstituted in 100 ul acidifedmethanol, adjusted to 1 ml with acidified water, and then partitionedagainst 2 ml hexane. After 30 min, the aqueous layer was isolated anddried as above. Dry samples were reconstituted in 800 ul acidiedmethanol and adjusted to 1 ml with acidied water. The reconstitutedsamples were passed through an equilibrated Sep-Pak C18 cartridges(Waters, Mississauga, ON, Canada), the final eluate was split in twoequal portions.

Hormone Quantification by HPLC-ESI-MS/MS

The procedure for quantification for abscisic acid and its metabolites,cytokinins, and gibberellins has been described in detail previously(Chiwocha 2003 2005). Samples were injected onto an ACQUITY UPLC HSS C18SB column (2.1×100 mm, 1.8 um) with an in-line filter and separate db agradient elution of water containing 0.02% formic acid against anincreasing percentage of a mixture of acetonitrile:methanol (50:50,v/v).

Briefly, the analysis using the Multiple Reaction Monitoring (MRM)function of the MassLynx 4.1 control software. The resultingchromatographic traces are quantified off-line by the software whereineach trace is integrated and the resulting ratio of signals(non-deuterated/internal standard) is compared with a previouslyconstructed calibration curve to yield the amount of analyze present (ngper sample). Results are expressed in nanograms per gram of dry weight.

Results

Induction of increased levels of ABA and its related metabolites by seedage is sufficient to prevent seed germination. FIG. 62 shows theincrease of gibberellin production, concomitant with the decreasedproduction of ABA and its related metabolites (shown in FIG. 63), bydirect and indirect application of SMCD 2206 in wheat. Germinationsuppression can be prevented by endophytes acting as inhibitors of ABAbiosynthesis, natural agents responsible for ABA degradation andinducers of gibberellin production. Further, lower cytokinin productionin the SMCD 2206 treatments (FIG. 64) is consistent with this mechanism.IAA-Asp concentration was higher in direct and indirect application ofSMCD 2206 as compared to the control (FIG. 65).

Example 21 Growth and Scale-Up of Microbes for Inoculation Growth andScale-Up of Bacteria for Inoculation on Solid Media

The bacterial isolates are grown by loop-inoculation of a single colonyinto R2A broth (supplemented with appropriate antibiotics) in 100 mLflasks. The bacterial culture is incubated at 30±2° C. for 2 days at 180rpm in a shaking incubator (or under varying temperatures and shakingspeeds as appropriate). This liquid suspension is then used to inoculateheat sterilized vermiculite powder that is premixed with sterile R2Abroth (without antibiotics), resulting in a soil like mixture ofparticles and liquid. This microbial powder is then incubated for anadditional couple of days at 30±2° C. with daily handshaking to aeratethe moist powder and allow bacterial growth. Microbially inoculatedvermiculite powder is now ready for spreading on to soil or onto plantparts. Alternatively, the R2A broth is used to inoculate Petri dishescontaining R2A or another appropriate nutrient agar where lawns ofbacteria are grown under standard conditions and the solid coloniesscraped off, resuspended in liquid and applied to plants as desired.

Growth and Scale-Up of Fungi for Inoculation on Solid Media

Once a fungal isolate has been characterized, conditions are optimizedfor growth in the lab and scaled-up to provide sufficient material forassays. For example, the medium used to isolate the fungus issupplemented with nutrients, vitamins, co-factors, plant-extracts, andother supplements that can decrease the time required to grow the fungalisolate or increase the yield of mycelia and/or spores the fungalisolate produces. These supplements can be found in the literature orthrough screening of different known media additives that promote thegrowth of all fungi or of the particular fungal taxa.

To scale up the growth of fungal isolates, isolates are grown from afrozen stock on several Petri dishes containing media that promotes thegrowth of the particular fungal isolate and the plates are incubatedunder optimal environmental conditions (temperature, atmosphere, light).After mycelia and spore development, the fungal growth is scraped andresuspended in 0.05 M Phosphate buffer (pH 7.2, 10 mL plate⁻¹).Disposable polystyrene Bioassay dishes (500 cm², Thermo Scientific NuncUX-01929-00) are prepared with 225 mL of autoclaved media with anyrequired supplements added to the media, and allowed to solidify. Platesare stored at room temperature for 2-5 days prior to inoculation toconfirm sterility. Five mL of the fungal suspension is spread over thesurface of the agar in the Bioassay plate in a biosafety cabinet, platesare allowed to dry for 1 h, and they are then incubated for 2-5 days, oruntil mycelia and/or spores have developed.

A liquid fungal suspension is then created via the following. Fungalgrowth on the surface of the agar in the Bioassay plates are thenscraped and resuspended in 0.05 M Phosphate buffer (pH 7.2). OD₆₀₀readings are taken using a spectrometer and correlated to previouslyestablished OD₆₀₀/CFU counts to estimate fungal population densities,and the volume adjusted with additional sodium phosphate buffer toresult in 100 mL aliquots of fungi at a density of approximately10⁶-10¹¹ spores mL⁻¹. This suspension may or may not be filtered toremove mycelia and can be used to create a liquid microbial formulationas described herein to apply the fungal isolate onto a plant, plantpart, or seed.

Growth and Scale-Up of Bacteria for Inoculation in Liquid Media

Bacterial strains are grown by loop-inoculation of one single colonyinto R2A broth (amended with the appropriate antibiotics) in 100 mLflasks. The bacterial culture is incubated at 28±2° C. for 1 day at 180rpm in a shaking incubator (or under varying temperatures and shakingspeeds as appropriate). The bacteria are pelleted by centrifugation andresuspended in sterile 0.1 M sodium phosphate. OD₆₀₀ readings are takenusing a spectrometer and correlated to previously established OD₆₀₀/CFUcounts to estimate bacterial population densities, and the volumeadjusted with additional sodium phosphate buffer to result in 100 mLaliquots of bacteria at a density of 1×10⁸ cells/mL. To help breaksurface tension, aid bacterial entry into plants and provide microbesfor some energy for growth, 10 μL of Silwet L-77 surfactant and 1 g ofsucrose is added to each 100 mL aliquot (resulting in 0.01% v/v and 1%v/v concentrations, respectively) in a similar way as in the protocolfor Agrobacterium-mediated genetic transformation of Arabidopsisthaliana seed [Clough, S., Bent, A. (1999) The Plant Journal 16(6):735-743].

Growth and Scale-Up of Fungi for Inoculation in Liquid Media

Once a fungal isolate has been characterized, conditions are optimizedfor growth in the lab and scaled-up to provide enough material forassays. For example, the medium used to isolate the fungi issupplemented with nutrients, vitamins, co-factors, plant-extracts,and/or other supplements that can decrease the time required to grow thefungal isolate and/or increase the yield of mycelia and/or spores thefungal isolate produces. These supplements can be found in theliterature or through screening of different known media additives thatpromote the growth of all fungi or of the particular fungal taxa.

To scale up the growth of fungal isolates, isolates are grown from afrozen stock on Petri dishes containing media that promotes the growthof the particular fungal isolate and the plates are incubated underoptimal environmental conditions (temperature, atmosphere, light). Aftermycelia and spore development, the fungal culture is scraped andresuspended in 0.05M Phosphate buffer (pH 7.2, 10 mL/plate). 1 liter ofliquid media selected to grow the fungal culture is prepared in 2 Lglass flasks and autoclaved and any required supplements added to themedia. These are stored at room temperature for 2-5 days prior toinoculation to confirm sterility. 1 mL of the fungal suspension is addedaseptically to the media flask, which is then incubated for 2-5 days, oruntil growth in the liquid media has reached saturation. Spore countswere determined using hemacytometer and correlated to previouslyestablished CFU counts to estimate fungal population densities, and thevolume adjusted with additional sodium phosphate buffer to result in 100mL aliquots of fungi at a density of approximately 10⁶-10¹¹ spores/mL.This suspension may or may not be filtered to remove mycelia and can beused to create a liquid microbial formulation as described herein toapply the fungal isolate onto a plant, plant part, or seed.

Creation of Liquid Microbial Formulations or Preparations for theApplication of Microbes to Plants

Bacterial or fungal cells are cultured in liquid nutrient broth mediumto between 10²-10¹² CFU mL⁻¹. The cells are separated from the mediumand suspended in another liquid medium if desired. The microbialformulation may contain one or more bacterial or fungal isolates. Theresulting formulation contains living cells, lyophilized cells, orspores of the bacterial or fungal isolates. The formulation may alsocontain water, nutrients, polymers and binding agents, surfactants orpolysaccharides such as gums, carboxymethylcellulose and polyalcoholderivatives. Suitable carriers and adjuvants can be solid or liquid andinclude natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, thickeners, binders orfertilizers. Compositions can take the form of aqueous solutions,oil-in-water emulsions, or water-in-oil emulsions. Small amounts ofinsoluble material can optionally be present, for example in suspensionin the medium, but it is generally preferred to minimize the presence ofsuch insoluble material.

Inoculation of Plants by Coating Microbes Directly onto Seed

Seed is treated by coating it with a liquid microbial formulation(prepared as described herein) including microbial cells and otherformulation components, directly onto the seed surface at the rate of10²-10⁸ microbial CFU per seed. Seeds are soaked in liquid microbialformulation for 1, 2, 3, 5, 10, 12, 18 or 24 hours or 2, 3, or 5 days.After soaking in microbial formulation, seeds are planted in growingcontainers or in an outdoor field. Seeds may also be coated with liquidmicrobial formulation by using an auger or a commercial batch treater.One or more microbial formulations or other seed treatments are appliedconcurrently or in sequence. Treatment is applied to the seeds using avariety of conventional treatment techniques and machines, such asfluidized bed techniques, augers, the roller mill method, rotostaticseed treaters, and drum coaters. Other methods, such as spouted beds mayalso be useful. The seeds are pre-sized before coating. Optionally themicrobial formulation is combined with an amount of insecticide,herbicide, fungicide, bactericide, or plant growth regulator, or plantmicro- or macro-nutrient prior to or during the coating process. Aftercoating, the seeds are typically dried and then transferred to a sizingmachine for grading before planting. Following inoculation, colonizationof the plants or seeds produced therefrom is confirmed via any of thevarious methods described herein. Growth promotion or stress resiliencebenefits to the plant are tested via any of the plant growth testingmethods described herein.

Inoculation of Plants with a Combination of Two or More Microbes

Seeds can be coated with bacterial or fungal endophytes. This methoddescribes the coating of seeds with two or more bacterial or fungalisolates. The concept presented here involves simultaneous seed coatingof two microbes (e.g., both a gram negative endophytic bacteriumBurkholderia phytofirmans and a gram positive endophytic bacteriumBacillus mojavensis). Optionally, both microbes are geneticallytransformed by stable chromosomal integration as follows. Bacillusmojavensis are transformed with a construct with a constitutive promoterdriving expression of a synthetic operon of GFPuv and spectinomycinresistance genes, while Burkholderia phytofirmans are transformed with aconstruct with a constitutive promoter driving expression of the lacoperon with an appended spectinomycin resistance gene. Seeds are coatedwith a prepared liquid formulation of the two microbes the variousmethods described herein. Various concentrations of each endophyte inthe formulation are applied, from 10² seed⁻¹ to about 10⁸ seed⁻¹.Following inoculation, colonization of the plants or seeds producedtherefrom may be confirmed via any of the various methods describedherein. Growth promotion or stress resilience benefits to the plant aretested via any of the plant growth testing methods described herein.

Example 22 In-Vitro Characterization of Endophytes

Endophytes may be characterized by their ability to produce certainsubstances. The following assays allow for the characterization ofendophytes.

Assay for Growth on Nitrogen Free LGI Media

All glassware is cleaned with 6 M HCl before media preparation. A new 96deep-well plate (2 mL well volume) is filled with 1 mL/well of sterileLGI broth (per L, 50 g Sucrose, 0.01 g FeCl₃-6H₂O, 0.8 g K₃PO₄, 0.2 gMgSO₄-7H₂O, 0.002 g Na₂MoO₄-2H₂O, pH 7.5). Bacteria are inoculated witha flame-sterilized 96-pin replicator. The plate is sealed with abreathable membrane, incubated at 25° C. with gentle shaking for 5 days,and OD₆₀₀, measurements are taken daily.

ACC Deaminase Activity Assay

Microbes are assayed for growth with ACC as their sole source ofnitrogen. Prior to media preparation all glassware is cleaned with 6 MHCl. A 2 M filter sterilized solution of ACC (#1373A, Research Organics,USA) is prepared in water. 1 μl/mL, of this is added to autoclaved LGIbroth (see above), and 1 mL aliquots are placed in a new 96 well plate.The plate is sealed with a breathable membrane, incubated at 25° C. withgentle shaking for 5 days, and OD_(600nm) readings are taken daily. Onlywells that are significantly more turbid than their correspondingnitrogen free LOT wells are considered to display ACC deaminaseactivity.

Mineral Phosphate Solubilization Assay

Microbes are plated on tricalcium phosphate media. This is prepared asfollows: 10 g/l. glucose, 0.373 g/L NH₄NO₃, 0.41 g/L MgSO₄, 0.295 g/LNaCl, 0.003 FeCl₃, 0.7 g/L Ca₃HPO₄ and 20 g/L Agar, pH 6, thenautoclaved and poured into 150 mm plates. After 3 days of growth at 25°C. in darkness, clear halos are measured around colonies able tosolubilize the tricalcium phosphate.

RNAse Activity Assay

1.5 g of torula yeast RNA (#R6625, Sigma) is dissolved in 1 mL of 0.1 MNa₂HPO₄ at pH 8, filter sterilized and added to 250 mL of autoclaved R2Aagar media which is poured into 150 mm plates. The bacteria from aglycerol stock plate are inoculated using a flame-sterilized 96 pinreplicator, and incubated at 25° C. for 3 days. On day three, plates areflooded with 70% perchloric acid (#311421, Sigma) for 15 minutes andscored for clear halo production around colonies.

Acetoin and Diacetyl Production Assay

1 mL of autoclaved R2A broth supplemented with 0.5% glucose is aliquotedinto a 96 deep well plate (#07-200-700. Fisher). The bacteria from aglycerol stock plate are inoculated using a flame-sterilized 96 pinreplicator, scaled with a breathable membrane, then incubated for 5 dayswith shaking (200 rpm) at 25° C. At day 5, 100 μl aliquots of cultureare removed and placed into a 96 well white fluorometer plate, alongwith 100 μl/well of Barritt's Reagents A and B which are prepared bymixing 5 g/L creatine mixed 3:1 (v/v) with freshly preparedalpha-naphthol (75 g/L in 2.5 M sodium hydroxide). After 15 minutes,plates are scored for red or pink colouration against a copper colourednegative control.

Auxin Production Assay

R2A agar media (Reasoner's 2A agar) supplemented with 5 mM L-tryptophanis autoclaved and poured into 150 mm plates. Using a 96 pin platereplicator, all microbes are inoculated onto the fresh plate from a 96well plate glycerol stock. The plate is incubated at 25° C. for 3 days,then overlaid with a nitrocellulose membrane, and put in a fridge at 4°C. overnight, allowing bacteria and their metabolites to infiltrate intothe paper. The next day, the nitrocellulose membrane is removed andplaced for 30 min on Whatman #2 filter papers saturated with Salkowskireagent (0.01 M ferric chloride in 35% perchloric acid, #311421, Sigma).Absorbance at 535 nm is measured using spectrophotometer. Auxinconcentration produced by bacterial isolates is determined usingstandard curves for IAA prepared from serial dilutions of 10-100 μgmL⁻¹.

Enzyme Production Assays

Oxidase and catalase activities are tested with 1% (w/v)tetramethyl-p-phenylene diamine and 3% (v/v) hydrogen peroxide solution,respectively. Gelatin and casein hydrolytic properties are analyzed bystreaking bacterial strains onto TSA plates from the stock culture.After incubation, trichloroacetic acid (TCA) is applied to the platesand an observation is made immediately for a period of at least 4 min(Medina and Baresi 2007, J Microbiol Methods 69:391-393). Chitinaseactivity of the isolates is determined as zones of clearing aroundcolonies following the method of Chernin et al. (1998) J Bacteriol180:4435-4441 (incorporated herein by rereference). Hemolytic activityis determined by streaking bacterial isolates onto Columbia 5% sheepblood agar plates. Protease activity is determined using 1% skimmed milkagar plates, while lipase activity is determined on peptone agar medium.Formation of halo zone around colonies was used as indication ofactivity (Smibert and Krieg 1994, In: Gerhardt P, Murray R, Wood W,Krieg N (Eds) Methods for General and Molecular Bacteriology, ASM Press,Washington, D.C., pp 615-640, incorporated herein by reference).Pectinase activity is determined on nutrient agar supplemented with 5 gL⁻¹ pectin. After 1 week of incubation, plates are flooded with 2%hexadecyl trimethyl ammonium bromide solution for 30 min. The plates arewashed with 1M NaCl to visualize the halo zone around the bacterialgrowth (Mateos et al. 1992, Appl Environ Microbiol 58:1816-1822,incorporated herein by reference).

Siderophore Production Assay

To ensure no contaminating iron is carried over from previousexperiments, all glassware is deferrated with 6 M HCl and water prior tomedia preparation. In this cleaned glassware, R2A agar media, which isiron limited, is prepared and poured into 150 mm Petri dishes andinoculated with bacteria using a 96 pin plate replicator. After 3 daysof incubation at 25° C., plates are overlaid with O-CAS overlay. 1 literof O-CAS overlay is made by mixing 60.5 mg of Chrome azurol S (CAS),72.9 mg of hexadecyltrimethyl ammonium bromide (HDTMA), 30.24 g offinely crushed Piperazine-1,4-bis-2-ethanesulfonic acid (PIPES) with 10mL of 1 mM FeCl₃.6H₂O in 10 mM HC solvent. The PIPES is finely powderedand mixed gently with stirring (not shaking) to avoid producing bubbles,until a dark blue colour is achieved. Melted 1% agarose is then added topre-warmed O-CAS just prior pouring the overlay in a proportion of 1:3(v/v). After 15 minutes, colour change is scored by looking for purplehalos (catechol type siderophores) or orange colonies (hydroxamatesiderophores).

Cellulase Activity Assay X

Adapting a previous protocol, 0.2% carboxymethylccllulose (CMC) sodiumsalt (#C5678, Sigma) and 0.1% triton X-100 (are added to R2A media,autoclaved and poured into 150 mm plates. Bacteria are inoculated usinga 96 pin plate replicator. After 3 days of culturing in the darkness at25° C., cellulose activity is visualized by flooding the plate withGram's iodine. Positive colonies are surrounded by clear halos.

Antibiosis Assay

Bacteria are inoculated using a 96 pin plate replicator onto 150 mmPetri dishes containing R2A agar, then grown for 3 days at 25° C. Atthis time, colonies of either E. coli DH5α (gram negative tester),Bacillus subtillus ssp. Subtilis (gram positive tester), or yeast strainAH 109 (fungal tester) are resuspended in 1 mL of 50 mM Na₂HPO₄ bufferto an OD₆₀₀ of 0.2, and 30 μl of this is mixed with 30 mL of warm LBagar. This is quickly poured completely over a microbe array plate,allowed to solidify and incubated at 37° C. for 16 hours. Antibiosis isscored by looking for clear halos around microbial colonies.

Assays for Exopolysaccharide, NH₃ and HCN Production

For exopolysaccharide (EPS) activity (qualitative), strains are grown onWeaver mineral media enriched with glucose and production of EPS isassessed visually (modified from Weaver et al. 1975, Arch Microbiol105:207-216). The EPS production is monitored as floc formation (fluffymaterial) on the plates after 48 h of incubation at 28° C. Strains aretested for the production of ammonia (NH₃) in peptone water as describedby Cappuccino and Sherman (1992), Biochemical activities ofmicroorganisms. In: Microbiology, A Laboratory Manual. TheBenjamin/Cummings Publishing Co. California, USA, pp 125-178,incorporated herein by reference. The bacterial isolates are screenedfor the production of hydrogen cyanide (HCN) by inoculating King's Bagar plates amended with 4.4 g L⁻¹ glycine (Lorck 1948, Physiol Plant1:142-146, incorporated herein by reference). Filter paper (Whatmanno. 1) saturated with picrate solution (2% Na₂CO₃ in 0.5% picric acid)is placed in the lid of a petri plate inoculated with bacterialisolates. The plates are incubated at 28±2° C. for 5 days. HCNproduction is assessed by the colour change of yellow filter paper toreddish brown.

Assays for Poly-Hydroxybutyrate (PHB) and n-Acyl-Homoserine Lactone(AHL) Production

The bacterial isolates are tested for PHB production (qualitative)following the viable colony staining methods using Nile red and Sudanblack B (Juan et al. 1998 Appl Environ Microbiol 64:4600-4602;Spiekermann et al. 1999, Arch Microbiol 171:73-80, each of which isincorporated by reference). The LB plates with overnight bacterialgrowth are flooded with 0.02% Sudan black B for 30 min and then washedwith ethanol (96%) to remove excess strains from the colonies. The darkblue coloured colonies are taken as positive for PHB production.Similarly, LB plates amended with Nile red (0.5 μL mL⁻¹) were exposed toUV light (312 nm) after appropriate bacterial growth to detect PHBproduction. Colonies of PHA-accumulating strains show fluoresce underultraviolet light. The bacterial strains were tested for AHL productionfollowing the method modified from Cha et al. (1998), Mol Plant-MicrobeInteract 11:1119-1129. The LB plates containing 40 μg ml⁻¹ X-Gal areplated with reporter strains (A. tumefaciens NTL4.pZLR4). The LB platesare spot inoculated with 10 μL of bacterial culture and incubated at28±2° C. for 24 h. Production of AHL activity is indicated by a diffuseblue zone surrounding the test spot of culture. Agrobacteriumtumefaciens NTL1 (pTiC58AaccR) is used as positive control and platewithout reporter strain is considered as a negative control.

Antagonistic Activities Against Plant Pathogenic Bacteria, Fungi andOomycetes

The antagonistic activities of bacterial isolates are screened againstplant pathogenic bacteria (Agrobacterium tumefaciens, Pseudomonassyringae, Streptococcus pneumoniae), fungi (Fusarium caulimons, Fusariumgraminarium, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani,Thielaviopsis basicola) and oomycetes (Phytophthora infestans,Phytophthora citricola, Phytophthora cominarum). For antibacterialassays, the bacterial isolates and pathogen are cultivated in trypticsoy broth at 30° C. for 24 h. The bacterial isolates are spot-inoculated(10 μL aliquots) on TSA plates pre-seeded with 100 μL tested pathogen.The plates are incubated at 28° C. for 48 h and clear zones ofinhibition are recorded.

Antagonistic activity of the bacterial isolates against fungi andoomycetes is tasted by the dual culture technique on potato dextroseagar (PDA) and yeast malt agar (YMA) media (Dennis and Webster 1971,Trans Brit Mycol Soc 57:25-39, incorporated herein by reference). Asmall disk (5 mm) of target fungus/oomycetes is placed in the center ofpetri dishes of both media. Aliquots of 10 μL of overnight bacterialcultures grown in tryptic soy broth are spotted 2 cm away from thecenter. Plates are incubated for 14 days at 24° C. and zones ofinhibition are scored.

Antagonistic activity of the fungal isolates against pathogenic fungiand oomycetes is tested by the dual culture technique on potato dextroseagar (PDA) and yeast malt agar (YMA) media (Dennis and Webster 1971,Trans Brit Mycol Soc 57:25-39, incorporated herein by reference). Asmall agar plug (5 mm in diameter) of target fungus/oomycetes is placednear the edge of petri dishes of both media, adjacent to an agar plug ofthe isolated fungus (as close as possible without touching). Plates areincubated for 14 days at 24° C. and radial growth or inhibition of it ismeasured.

While the present disclosure has been described with reference to whatare presently considered to be the examples, it is to be understood thatthe disclosure is not limited to the disclosed examples. To thecontrary, the disclosure is intended to cover various modifications andequivalent arrangements included within the spirit and scope of theappended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1  Sequences 2204 ITS rDNACCTATAGCTGACTGCGGAGGGACATTACAAGTGACCCCGGTCTAACCACCGGGATGTTCATAACCCTTTGTTGTCCGACTCTGTTGCCTCCGGGGCGACCCTGCCTTCGGGCGGGGGCTCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGCAGTCTGAGTAAACTTAATTAATAAATTAAAACTTTTAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCACCACTCAAGCCTCGCTTGGTATTGGGCAACGCGGTCCGCCGCGTGCCTCAAATCGACCGGCTGGGTCTTCTGTCCCCTAAGCGTTGTGGAAACTATTCGCTAAAGGGTGTTCGGGAGGCTACGCCGTAAAACAACCCCATTTCTAAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCCCCAGTAACGAA (SEQ ID NO: 1)>Sarocladium sp. SMCD 2204F LSU rRNACAATGGGGAGTGTCGTCTTCTAAGCTAAATACCGGCCAGAGACCGATAGCGCACAAGTAGAGTGATCGAAAGATGAAAAGCACTTTGAAAAGAGGGTTAAAAAGTACGTGAAATTGTTGAAAGGGAAGCATTCATGACCAGACTTGGGCTTGGTTGAACATCCGGCGTTCTCGCCGGTGCACTCTGCCAGTCCAGGCCAGCATCAGTTTGCCCCGGGGGACAAAGGCGGTGGGAATGTGGCTCCCTTCGGGGAGTGTTATAGCCCGCCGTGTAATGCCCTGGGGCGGACTGAGGAACGCGCTTCGGCACGGATGCTGGCGTAATGGTCATCAATGACCCGTCTTGAAACACGGACCAAGGAGTCTAACATCA(SEQ ID NO: 2) >2206 ITS rDNATCGACGGCGTATCCTAGTGACTGCGGAGGATCATTACCGAGTGAGGGCCCTCTGGGTCCAACCTCCCACCCGTGTTTAATTTACCTTGTTGCTTCGGCGGGCCCGCCTTAACTGGCCGCCGGGGGGCTTACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAACTCTGTCTGAAGATTGTAGTCTGAGTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCACGGCTTGTGTGTTGGGCCCCGTCCTCCGATCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA (SEQ ID NO: 3) >2208 ITS rDNATAACTGATTTGGCGGACTGGCGGAAGGACATTAAAGAGACGTTGCCCTTCGGGGTATACCTCCCACCCTTTGTTTACCTTTTCCTTTGTTGCTTTGGCGGGCCCGTCCTCGGACCACCGGTTTCGGCTGGTCAGTGCCCGCCAGAGGACCTAAAACTCTGTTTGTTCATATTGTCTGAGTACTATATAATAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTACAACCCTCAAGCTCTGCTTGGTATTGGGCTCTGCCGGTCCCGGCAGGCCTTAAAATCATTGGCGGTGCCATTCGGCTTCAAGCGTAGTAATTCTTCTCGCTTTGGAGACCCGGGTGCGTGCTTGCCATCAACCCCCAATTTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGTCCCAATAACGAATTTATAAATAATA (SEQ ID NO: 4) >2210 ITS rDNATCGAGAGTTCGGACTAAGTGCCTGATCCGAGGTCAAGACGGTAATGTTGCTTCGTGGACGCGGGCCACGCCCCCCCGCAGACGCAATTGTGCTGCGCGAGAGGAGGCAAGGACCGCTGCCAATGAATTTGGGGCGAGTCCGCGCGCGAAGGCGGGACAGACGCCCAACACCAAGCAGAGCTTGAGGGTGTAGATGACGCTCGAACAGGCATGCCCCATGGAATACCAAGGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACACTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAGCCAAGAGATCCATTGTTGAAAGTTGTAACGATTGTTTGTATCAGAACAGGTAATGCTAGATGCAAAAAAGGTTTTGTTAAGTTCCAGCGGCAGGTTGCCCCGCCGAAGGAGAACGAAAGGTGCTCGTAAAAAAAGGATGCAGGAATGCGGCGCGTGAGGGTGTTACCCCTACCACCCGGGAGAGAACCCCCGAGGGCCGCGACCGCACCTGGTTGAGATGGATAATGATCCTTCCGCAGGTTCACCTACGGAAACC (SEQ ID NO: 5) >2215 16S rDNACCGGGGGCACTCCACTGCGTATGTGTGACGAGTAGACCGCTGCGCTTAGCTGAGGTCTGATGAAATGTAGAACACTTAACAAAAATATGCCCGGATGGATATACTTTTCAACGACAGGGCTGCGATTGGATGATCTCCTTTGAAACACAGAACTAGTCACGGCGACGAATACTCAACTTCGACCCCCCCCCTTTCTGGAGGCGCGTCTTAGTCCCCTCCTTGATGGAGCTGCCCCGTGCTCGGCGGCCGGAGTCGGCGGTGTTTTCCGCTGTACCTGAGACGCTGGACCAACTCCTTCGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGATTGTGAAAGCCCGAGGCTTAACCTCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTGTGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAACATCCAGAGATGGGTGCCCCCTTGTGGTCGGCGTACAGGTCGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTAAGTCCCGCAACGAGCGCAACCTTGTTCTGGTGCTGCCAGCATGCCCTTCGGGTGATGGGACTTCACCACGGAGACCGCGGCTCCACTCCGACGAGGTGGGGGACGACGTCAGTCATCATGCCCTAATGTCTGGCTG (SEQ ID NO: 6)

TABLE 2 SMCD endophytic root colonization frequency assessed in 3D wheatgerminant radicles. Endophytes SMCD2204 SMCD2206 SMCD2210 SMCD2215 %colonization 43 40 49 48

TABLE 3 Energy of germination (EG) and hydrothermal time (HTT) of wheatseeds grown under heat (36° C.), drought (potato dextrose agar (PDA)media plus 8% polyethylene glycol (PEG) 8000), heat and drought combinedand control in vitro conditions. 1. Heat Drought Heat and DroughtControl HTT to 50% HTT to 50% HTT to 50% HTT to 50% germinationgermination germination germination (MPa ° C. (MPa ° C. (MPa ° C. (MPa °C. Endophyte EG (days) days EG (days) days) EG (days) days) EG (days)days) SMCD 2204 3.7 ± 0.3 91 ± 7 2.9 ± 0.3 52 ± 5 2.0 ± 0.8 22 ± 8 1.6 ±0.2 65 ± 8 SMCD 2206 2.5 ± 0.3 62 ± 7 1.9 ± 0.1 * 34 ± 2 * 2.0 ± 0.8 22± 8 1.5 ± 0.2 61 ± 8 SMCD 2208 3.7 ± 0.3 91 ± 7 3.0 ± 0.3 53 ± 5 4.0 ±1.0 43 ± 10 1.6 ± 0.2 65 ± 8 SMCD 2210 1.8 ± 0.2 * 44 ± 5 * 2.2 ± 0.2 *39 ± 3 * 1.0 ± 0.5 11 ± 5 1.6 ± 0.2 65 ± 8 SMCD 2215 2.5 ± 0.3 62 ± 72.3 ± 0.2 * 41 ± 3 * 1.3 ± 0.2 14 ± 2 1.5 ± 0.2 61 ± 8 No Endo 3.8 ± 0.594 ± 11 4.5 ± 0.5 80 ± 8 3.0 ± 1.5 32 ± 15 1.6 ± 0.2 65 ± 8 Within acolumn, data followed by an asterisk (*) are significantly differentfrom the no endophyte control (p ≦ 0.05; ANOVA, followed by a post-hocLSD test). Note: The seeds used in EG and HTT determination were fromthe second round of experiments, and hence subjected to sterilization in5% sodium hypochlorite for one minute, rather than three;SMCD—Saskatchewan Microbial Collection and Database

TABLE 4 Endophytes increase drought tolerance efficiency (DTE) and yieldin barley and wheat under stress conditions. Control conditions DroughtStress Average YIELD Average YIELD spikes g spikes g DTE^(‡)(3plants/pot) Increased (3plants/pot) Increased Crop Genotypes (%) E− E+% E− E+ % AC Avonlea 16.1 18.27 25.52 28.41 2.94 10.62 72.32 (Cont) PT580 57.3 23.42 32.60 28.16 13.38 21.53 37.85 Control CDC Utmost 72.320.55 35.4 41.95 16.67 29.8 44.06 VB Strongfield 75.6 13.54 16.77* 19.2610.23 14.98 31.71 WHEAT Unity VB 75.3 20.72 26.6 22.11 15.61 23.2 32.72CDC Teal 76.9 19.51 30.37 35.76 14.90 25.1 40.64 Carberry 83.8 17.3133.07 47.66 14.52 22.9 36.59 BW 423 85.0 13.26 25.83 48.66 12.28 21.4142.64 CDC Veronna 87.8 15.35 22.58 32.02 13.49 20.16 33.09 Lillian 87.820.50 28.3 27.56 18.1 23.6 23.31 Two row barley CDC Copeland 4.9 6.0110.78 44.25 2.91 6.95 58.13 CDC Kendall 13.2 9.93 24.19 58.95 0.32 1.0368.93 BARLEY AC Metcalfe 43.2 16.5 22.4* 26.34 7.3 14.05 48.04 New Dale72.1 9.55 26.88 64.47 6.89 12.17 43.39 Six row barley Legacy 1.1 20.4226.87* 24.00 2.26. 2.38* 5.04 CDC Bold 57.0 9.16 19.9 53.97 5.22 7.530.40 ^(‡)Drought tolerance efficiency (DTE) = (Yield under stress/Yieldunder non-stress) × 100; presented in increasing order within the Table.Genotypes with high DTE are considered as drought resistant; whereasgenotypes with low DTE are considered as drought susceptible. Note:Effect of the endophyte's absence (E−) or presence (E+) on genotypeyield was calculated as an average of all three tested SMCD 2206, SMCD2210, and SMCD 2215 strains. *Within the rows, a mean is notstatistically significant at p ≧ 0.05.

TABLE 5 Rhizobium sequence maximum identity against GenBank database MaxTotal Query E Max Accession Description score score coverage value identEF549401.1 Rhizobium sp. CCBAU 83431 16S 1007 1007 46% 0.0 99% ribosomalDNA gene, partial sequenceNative Rhizobium Nodulator in Interaction with Streptomyces SMCD2215

16S F (Golden) Rhizobium sp. (SEQ ID NO: 7)GGAAGGGGGGCGGCTTACCATGCAAGTCGAGCGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCCTTGACTACGGAATAACGCAGGGAAACTTGTGCTAATACCGTATGTGTCCTTCGGGAGAAAGATTTATCGGTCAAGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCACCGGAGAAGATAATGACGGTATCCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGCCGTAAAGCGCACGTAGGCGGATCGATCAGTCAGGGGTGAAATCCCAGGGCTCAACCCTGGAACTGTCTTTGATACTAGCACCGGCACCCCGTTTCGACATGCAAAAAATGATGCCCAGGCTTATGTGTCGATCTGGAGAACTTCCTGCTCGAGTGATTTACCCACATGGCGTTGAACCTGGCTGCTACGGCTCTCTTCGGCGTGGACCCCGGCCTCCTATCCCCGGGATGCCACCCATGGACGCCGCAGTCTCCATGGATATATCATGGAGGTGGGTTTTCTCCGACTCATGATGCCGGCTTCTTGCTGGAAGTTGATGAAGCAACTAAACATCAGCCCTGAGAGAAAGCTTCGCATGCCGCGCAGGGTGCTCCGAGTGTTCGTCTGGAGATGATGAAATAGACGAAGATCATCTCATGTCATGTTGGTAACGACGAGAACAAGATGGTGTGGATTTTGTGTCTTCCATCCTCCATGACCCTGACGATGCTGATGATGACGTGGTTCATGCTATGATGACTCGATACTGGTCGCTGCAAGCGGATACAGTTGGGACCTACCGCTAACATGGTTCTTTCTACAACCTCCCCCCAAACCGCATAGGATCGTGGTCAATCATTCGGCACGAACCTCTTCCCCCATTGCCTCCAACTAGTTTATCGCTCTAGAGTTGGGGAGCCCTGTGTGACCTTTCGTACGCGA 

TABLE 6  Set of SOD, MnSOD and Pro primers used to assess pea [Handel]genes expression exposed to PEG drought/osmotic stress by qPCR Gene NamePrimer Reference PP2A CCACATTACCTGTATCGGATGACA (F)Die et. al, Planta (2010) internal (SEQ ID NO: 8) 232:145-153 controlGAGCCCAGAACAGGAGCTAACA (R) (SEQ ID NO: 9) MnSOD saltgcagaaaaaccctatcctccgtgct (F) Wong Vega et. al., Plant and drought(SEQ ID NO: 10) Mol. Biol. 17 (6), 1271- gctccaaagctccgtagtcg (R)1274 (1991) (SEQ ID NO: 11) Pea SOD ctgtactcgctgttggggtg (F)Nakamura et. al., Plant (SEQ ID NO: 12) Biotechnol. 20, 247-253gcatggatatggaagccgtg (R) (2003) (SEQ ID NO: 13) Proline (Pro)aatggccgaaagcattgcca (F) Williamson, C.L. and (SEQ ID NO: 14)Slocum, R.D., Plant aaggacggtgatgccgatggactc (R) Physiol. 100, 1464-1470 (SEQ ID NO: 15) (1992)

TABLE 7 Evaluation of the efficiency of seed sterilization methods.Seeds were germinated on potato dextrose agar for 4 days at ambienttemperature (20° C.). Each petri dish had 10 wheat seeds. Potatodextrose agar (PDA) Sterilization type Contamination Germination Control50% 80% 50% Bleach 0 50% 95% Ethyl alcohol 0 70% 50% Bleach + 95% 0 50%Ethyl alcohol Chlorine gas 0 80%

TABLE 8 Average germination of wheat seeds under cold and biologicalstratification treatments Cold Endophyte- Endophyte Day ControlStratification indirect direct 1 6.66 ± 6.66^(ab) 16.6 ± 3.33^(ab) 0.00± 0.00^(a) 26.6 ± 12.02^(b) 2 16.6 ± 8.81^(p) 40.0 ± 11.5^(pq) 50.0 ±5.77^(q) 66.6 ± 8.81^(q) 3 33.3 ± 12.01^(x) 53.3 ± 8.81^(xy) 73.3 ±3.33^(yz) 86.9 ± 7.24^(z) * Duncan test was performed to testsignificant difference among the treatments (Control, ColdStratification, Endophyte-indirect, and Endophyte direct) on Day 1 (a,b, c), Day 2 (p, q), and Day 3 (x, y, z) ** Different letters indicatesignificant difference at P < 0.05

TABLE 9  Transcription factor Myb2 mRNA (158 bp) TaMyb2 1Facatcaagcgcggcaacttca (SEQ ID NO: 16) TaMyb2 1Rgagccgcttcttgaggtgggtgt (SEQ ID NO: 17)Transcription factor Myb1 mRNA (152 bp) TaMyb1 1Fccagggaggacggacaacga (SEQ ID NO: 18) TaMyb1 1Rctctgcgccgtctcgaagga (SEQ ID NO: 19)

TABLE 10 Summary of the crops tested in the field and their respectivevarieties Crop Varieties Tested Corn 39A16, 40R73 Spring Wheat Lillian,Unity, Utmost, Briggs, Prosper, Select Durum Wheat Strongfield BarleyBold, Kendal Canola Victory 1, Victory 2 Pea Meadow (green), Striker(yellow) Chickpea Counsul, Frontier Lentil Dazil, Impower

TABLE 11 Summary of the targeted seeding density, planting date, andharvesting date for each crop and location. Targeted Seeding PlantingHarvesting Location Crop Density Date* Date* Saskatchewan, Spring 4200Jun 24/ Sep 18/ Canada Wheat seeds/15 m³ June 25 Sep 24 Durum 4200 Jun24/ Sep 18/ seeds/15 m³ June 25 Sep 24 Barley 5400 Jun 24/ Sep 18/seeds/15 m³ June 25 Sep 24 Canola 1600 Jun 23/ Sep 18/ seeds/15 m³ Jul 4Sep 24 Pea 3200 Jun 23/ Sep 18/ seeds/15 m³ Jul 4 Sep 24 Chickpea 6500Jun 24/ Sep 18/ seeds/15 m³ Jul 4 Sep 24 Lentil 2000 Jun 24/ Sep 18/seeds/15 m³ Jul 3 Sep 24 Brookings, Spring 65 lb- May 14 Sep 9 SouthDakota Wheat seed/acre Corn 32,500 Jun 5 Nov 14 seeds/acre York,Nebraska Corn 32,500 Jun 19 Nov 14 seeds/acre *indicates first plantingand harvesting dates were for Vanguard and second were for StewartValley.

TABLE 12 The abbreviation and full name of the Gibberellin derivativesGA1 Gibberellin 1 GA19 Gibberellin 19 GA44 Gibberellin 44 GA53Gibberellin 53

TABLE 13 The abbreviation and full name of abscisic acid and its relatedmetabolites ABA cis-Abscisic acid ABAGE Abscisic acid glucose ester PAPhaseic acid 7′OH-ABA 7′-Hydroxy-abscisic acid t-ABA trans-Abscisic acid

TABLE 14 The abbreviation and full name of the cytokinins and itsrelated metabolites c-ZOG trans-Zeatin-O-glucoside c-Z cis-Zeatin C-ZRczs-Zeatin riboside

REFERENCES

-   Abdellatif et al. 2009. Mycological Research, 113:782-791.-   Abdellatif et al. 2010. Can J Plant Pathol, 32: 468-480.-   Adriaensen et al. 2006. Mycorrhiza, 16: 553-558.-   Agius et al. 2006. PNAS, 103: 11796-11801.-   Ali et al. 1994. Annals of Applied Biology, 125: 367-375.-   Allen 1958. Forest Chron, 34: 266-298.-   Armas et al. 2004. Ecology, 85: 2682-2686.-   Arnold et al. 2001. Mycological Research, 105: 1502-1507.-   Bacon and White 2000. In: Bacon CW and White JFJ (Eds), Microbial    endophytes. Marcel Dekker Inc; New York, N.Y., USA. 237-263.-   Bae et al. 2009. J Exp Bot 60: 32793295.-   Baird et al. 2010. Mycorrhiza. 20: 541-549.-   Barrero et al. 2009. Plant Physiology, 150: 1006-1021.-   Baskin et al. 1992. International Journal of Plant Sciences, 153:    239-243.-   Baskin and Baskin 2004. Sci. Res., 14: 1-16.-   Bewley and Black 1982. Physiology and Biochemistry of Seeds. 2.    Viability, Dormancy, and Environmental Control. Springer-Verlag,    Berlin.-   Bloom and Richard 2002. ASAE Paper No 027010. ASAE, St. Joseph,    Mich.-   Bogatek and Lewak 1988. Physiologia Plantarum, 73: 406-411.-   Boyko and Kovalchuk 2008. Environmental and Molecular Mutagenesis,    49: 61-72.-   Bradford 2002. In: J. Kigel J, Galili G (eds), Seed Develop and    Germin. Marcel Dekker Inc, New York, pp. 351-396.-   Calcagno et al. 2012. Mycorrhiza, 22:259-69.-   Cao and Moss. 1989. Crop Sci, 29: 1018-1021.-   Carpita et al. 1983. Physiologia Plantarum 59: 601-606.-   Cavieres and Arroyo, 2000. Plant Ecology 149: 1-8.-   Cavieres and Arroyo, 2000b. Gayana Botanica 64: 40-45.-   Charlton et. al. 2008. Metabolomics, 4: 312-327.-   Chau et al. 2012. Fungal Biology, 116:1212-1218.-   Chipanshi et al. 2006. Clim Res, 30: 175-187.-   Chiwocha et al. 2003. Plant J., 3:405-417.-   Chiwocha et al. 2005. Plant J., 42:35-48.-   Choi and Sano, 2007. Molecular Genetics and Genomics, 277: 589-600.-   Davitt et al. 2010. New Phytol, 188: 824-834. de Bary 1866. Vol. II.    Hofmeister's Handbook of Physiological Botany. Leipzig, Germany.-   Desfeux et al., 2000. Plant Physiology, 123: 895-904.-   Dong-dong et al. 2009. J Zhejiang Univ-Sci B, 9: 964-968.-   Farquhar and Richards 1984. Australian Journal Plant Physiology 11:    539-552.-   Farquhar et al. 1989 In: Jones H G, Flowers T J and Jones M B (Eds)    Plants under stress.-   Cambridge University Press, Cambridge, pp 47-69.-   Farquhar et al. 1989b. Annual Review of Plant Physiology and Plant    Molecular Biology. 40: 503-537.-   Finnegan et al. 1998. Plant Molecular Biology 95: 5824-5829.-   Freeman 1904. Philosophical Transactions of the Royal Society London    (Biology) 196: 1-27.-   Friend et al. 1962. Can J Bot, 40: 1299-1311.-   Gan et al. 2004. Can. J Plant Sci, 84: 697-704.-   Germida et al. 2010. Field-scale assessment of phytoremediation at a    former oil tank battery in Bruderheim, Alberta. World Congress of    Soil Science, Soil Solutions for a Changing World, 1-6 Aug. 2010.    Brisbane, Australia. Available on-line at: http://www.iuss.org/19th    %20WCSS/Symposium/pdf/0694.pdf-   Gizinger 2002. Experimental Hematology, 30: 503-512.-   Gornall et al. 2010. Phil. Trans. R. Soc. B, 365, 2973-2989.-   Grant et al. 2009. Tree Physiology, 29: 1-17.-   Gummerson 1986. J Exp Bot, 37: 729-741.-   Gundel et al. 2010. Evol Appi, 3: 538-546.-   Guo et al. 2003. Science, 302: 100-103.-   Hayat et al. 2010. Nitric Oxide in Plant Physiology, Issue 58,    Willey-VCH Verlag, Germany.-   Hedden and Phillips, 2000. Trends in Plant Science, 5: 523-530.-   Hubbard et al. 2011. In: Wheat: Genetics, Crops and Food Production.    Nova Science Publishers Hauppauge, N.Y., USA. pp. 333-345.-   Hubbard et al. 2012. Botany, 90(2): 137-149.-   Jame et al. 1998. Agric Forest Meteorol 92: 241-249.-   Ji et al., 2011. Plant Physiology, 156: 647-662.-   Johannes et al. 2009. Plos Genetics 5: e1000530.-   Johannes et al. 2011. Genetics, 188: 215-227.-   Johnson et al. 1990. Crop Science, 30: 338-343.-   Jost et al. 2001. Nucleic Acids Research, 29: 4452-4461.-   Jumpponen and Trappe 1998. New Phytologist, 140: 295-310.-   Jurado et al., 2010. Food Microbiology, 27: 50-57.-   Kane 2011. Environmental and Experimental Botany, 71: 337-344.-   Kang et al. 2008. International Journal of Sustainable Development    and World Ecology, 15: 440-447.-   Karavata & Manetas 1999. Photosynthetica, 36: 41-49.-   Khan et al. 2010. Pakistan Journal of Botany, 42: 259-267.-   Khan et al. 2012. BMC Microbiol, 12; 12:3.-   Kiffer and Morelet 2000. Science Publisher Inc, Enfield, N.H.,    Plymouth.-   Kochy and Tielborger 2007. Basic Appl Ecol 8: 171-182.-   Koyuncu 2005. Acta Biologica Cracoviensia Series Botanica, 47:    23-26.-   Labeda et al 2012. Antonie van Leeuwenhoek, 101:73-104.-   Lang-Mladek et al. 2010. Molecular Plant, 3: 594-602.-   Larran et al. 2002. World Journal of Microbial Biotechnology, 18:    683-686.-   Leone et al. 1994. Physiol Plantarum, 92: 21-30.-   Li et al. 2008. Ecological Research, 23: 927-930.-   Li et al. 2011. Agronomy Journal, 103: 1619-1628.-   Lu et al. 2007. Plant Biology, 49: 1599-1607.-   Lucht et al. 2002. Nature Genetics, 30: 311-314.-   Madsood et al. 2005. Engineering Applications of Artificial    Intelligence, 18: 115-125.-   Margulis, 1991. In Symbiosis as a Source of Evolutionary    Innovation, L. Margulis and R. Fester, ed. The MIT Press: Cambridge.    pp. 1-14.-   Marquez et al. 2007. Science, 315: 513-515.-   McCormick M C, Siegel (eds.) 1999. Prenatal Care: Effectiveness and    implementation.-   Cambridge University Press UK.-   McDonald 2009. Handbook of biological statistics. 2nd ed. Sparky    House Publishing, Baltimore, Md.-   McMaster 2009. In: Carver BF (ed), Wheat, science and trade,    Wiley-Blackwell, Iowa, USA, pp. 31-55.-   Milberg and Andersson 1998. Plant Ecology, 134: 225-234.-   Millar et al., 2006. Plant Journal, 45: 942-954.-   Miransari et al. 2011. Applied Microbiology and Biotechnology, 92:    875-885.-   Mitchell et al., 2009. Microbiology-SGM, 156: 270-277.-   Mühlmann and Peintner 2000. Mycorrhiza, 18: 171-180.-   Mukhopadhyay et al., 2004. PNAS, 101: 6309-6314.-   Nakatsubo et al. 1998. FEBS Lett, 427:263-266.-   Nakamura et al., 2010. Euphytica, 171: 111-120.-   Nelson, 2004. Annu Rev Phytopathol, 42: 271-309.-   Nicot 2005. Journal of Experimental Botany, 56: 2907-2914.-   Nonogaki et al., 2010. Plant Science, 179: 574-581.-   Oikawa et al., 2004. Plant Molecular Biology, 55: 687-700.-   Okamoto et al., 2006. Plant Physiology, 141:97-107.-   Oliver et al., 2007. Plant and Cell Physiology, 48: 1319-1330.-   Penterman et al. 2007. PNAS, 104: 6752-6757.-   Phillips et al., 1995. Plant Physiology, 108: 1049-1057.-   Probert et al., 1989. Journal of Experimental Botany, 40: 293-301.-   Qin and Zeevart, 1999. PNAS, 96: 15354-15361.-   Reynolds et al. 2007 Journal of Experimental Botany, 58: 177-186.-   Richards et al. 2002. Crop Science, 42: 111-121.-   Ries et al. 2000. Nature, 406: 98-101.-   Rivero et al. 2011. International Conference on Arabidopsis    Research. June 22-25, Madison USA.-   Ruan et al. 2002. Seed Sci Technol, 30: 61-67.-   Ryan et al. 2008. FEMS Microbiol Lett, 278: 1-9.-   Saikkonen et al., 1998. Annual Review of Ecology and Systematics,    29: 319-343.-   Saze 2008. Seminars in Cell and Developmental Biology, 19: 527-536.-   Schrey and Tarkka 2008. Antonie van Leeuwenhoek, 94:11-19.-   Schutz and Rave 1999. Ecology, 144: 215-230.-   Semenov and Shewry 2011. Scientific Reports, 1: 66-71.-   Sinclair et al. 1984. BioScience, 34: 36-40.-   Singh et al. 2011. Plant Signal Behav, 6: 175-191.-   Smith and Read 2008. Mycorrhizal symbiosis, Third Edition. Elsevier    Ltd. Mycorrhizas in acholorophyllous plants (mycoheterotrophs).    Chapter 13: 458-507.-   Solaiman et al. 2010. Australian Journal of Soil Research, 48:    546-554.-   Soleimani et al. 2010. Chemosphere, 81: 1084-1090.-   Stone et al., 2000. In: Bacon, C. W. and White, J. F. eds.,    Microbial Endophytes, Marcel Dekker: New York Chap. 1: 3-29.-   Strobel et al., 2004. Journal of Natural Products, 67: 257-268.-   Sun et al. 2010. Journal of Plant Physiolog, 167: 1009-1017.-   Tan and Zou, 2001. Nat Prod Rep, 18: 448-45.-   Tokala et al. 2002. Appl Environ Microbiol, 68:2161-2171.-   Vaughn et al. 2007. PloS Biology, 5: 1617-1629.-   Verhoeven et al. 2010. New Phytologis, 185: 1108-1118.-   Vujanovic et al. 2000. Annals of Botany, 86: 79-86.-   Vujanovic and Brisson 2002. Mycological Progress. 1: 147-154.-   Vujanovic and Vujanovic 2006. Floriculture, Ornamental and Plant    Biotech, 63: 563-569.-   Vujanovic and Vujanovic 2007. Symbiosis, 44: 93-99.-   Vujanovic 2007b. Can J Plant Pathol, 29: 451-451.-   Vujanovic 2008. 19th International Conference on Arabidopsis.    Research Proceedings—ICAR13,-   July 23-27, Montreal, QC, Canada.-   Waller et al. 2005. PNAS, 102: 13386-13391.-   Wallin 1927. Symbionticism and the Origin of Species. London:    Baillière, Tindall and Cox.-   Wang et al. 2011. Journal of Experimental Botany, 62: 1951-1960.-   Whalley et al. 2006. Plant and Soil, 280: 279-290.-   White and Torres 2010. Physiol. Plant, 138: 440-446.-   Wu et al. 2008. Plant Physiology, 148: 1953-1963.-   Wu and Sardo 2010. Lichtfouse E. (Ed.), Sociology, Organic Farming,    Climate Change and Soil Science. Sustainable Agriculture Reviews. 3:    DOI 10.1007/978-90-481-3333-8_(—)3.-   Yamaguchi et al. 1998. Plant Cell, 10: 2115-2126-   Yang et al., 2002. Planta, 215: 645-652.-   Zadoks et al. 1974. Weed Research, 14:415-421.-   Zhang et al., 2007. BMC Genet, 2007, 8: 40.-   Zhang et al. 2010. Journal of Cereal Science, 52: 263-269.-   Zhang et al. 2011. African Journal of Microbiology Research, 5:    5540-5547.-   Zhao et al. 2007. Journal of Plant Nutrition, 30: 947-963.-   Zhong et al. 2009. African Journal of Biotechnology, 8: 6201-6207.-   Zhu et al. 2007. Current Biology, 17: 54-59.-   Foresight. The future of food and farming: challenges and choices    for global sustainability. Final Project Report. London: The    Government Office for Science, UK, 2011.-   IPCC Climate Change 2007. In: Solomon S, Qin D, Manning M, Chen Z,    Marquis M, Averyt K B, Tignor M and Miller H L (Eds). Cambridge    University Press, Cambridge, UK.-   Saskatchewan Ministry of Agriculture 2008. Varieties of Grain Crops.    SaskSeed guide. Regina, SK, Canada

1. A synthetic preparation comprising an agricultural plant seed and acomposition comprising an endophyte capable of promoting germination andan agriculturally-acceptable carrier, wherein an agricultural plantgrown from the seed has an altered trait as compared to a controlagricultural plant.
 2. The synthetic preparation of claim 1, wherein theendophyte capable of promoting germination is a coleorhiza-activatingendophyte and the agricultural plant seed is a monocot seed.
 3. Thepreparation of claim 1, wherein said composition is disposed on anexterior surface of the agricultural seed in an amount effective tocolonize the cortical cells of an agricultural plant grown from the seedand to produce the altered trait, wherein the altered trait is animproved functional trait selected from the group consisting ofincreased yield, faster seedling establishment, faster growth, increaseddrought tolerance, increased heat tolerance, increased cold tolerance,increased salt tolerance, increased tolerance to Fusarium infection,increased tolerance to Puccinia infection, increased biomass, increasedroot length, increased fresh weight of seedlings, increased plant vigor,nitrogen stress tolerance, enhanced Rhizobium activity, enhancednodulation frequency, and early flowering time.
 4. The preparation ofclaim 3, wherein said composition is disposed on an exterior surface ofthe agricultural seed in an amount effective to colonize at least 1% ofthe cortical cells of an agricultural plant grown from the seed.
 5. Thepreparation of claim 1, wherein said composition is disposed on anexterior surface of the agricultural seed in an amount effective tocause a population of seeds inoculated with said composition to havegreater germination rate, faster dormancy breakdown, increased energy ofgermination, increased seed germination vigor or increased seed vitalitythan a population of control agricultural seeds.
 6. The preparation ofclaim 5, wherein said composition is disposed on an exterior surface ofthe agricultural seed in an amount effective to cause a population ofseeds inoculated with said composition to reach 50% germination fasterthan a population of control agricultural seeds.
 7. The preparation ofclaim 1, wherein the endophytes are a selected from the group consistingof a spore-forming endophyte, a facultative endophyte, a filamentousendophyte, an endophyte capable of living within another endophyte, anendophyte capable of forming hyphal coils within the plant, an endophytecapable of forming microvesicles within the plant, an endophyte capableof forming micro-arbuscules within the plant, an endophyte capable offorming hyphal knots within the plant, an endophyte capable of formingHartig-like nets within the plant, and an endophyte capable of formingsymbiosomes within the plant.
 8. The preparation of claim 1, whereinsaid composition is disposed on an exterior surface of the agriculturalseed in an amount effective to colonize the cortical cells of anagricultural plant grown from the seed and to produce the altered trait,wherein the altered trait is altered gene expression, wherein the geneis selected from the group consisting of a gene involved in plantgrowth, an acquired resistance gene, and a gene involved in protectionfrom oxidative stress.
 9. The preparation of claim 8, wherein the geneis a gene involved in phytohormone production.
 10. The preparation ofclaim 8, wherein the gene is a redox-regulated transcription factor. 11.The preparation of claim 8, wherein the gene is a gene involved insuperoxide detoxification or in NO production or breakdown.
 12. Thesynthetic preparation of claim 1, wherein the agricultural plant seed isselected from the group consisting of corn, soy, wheat, cotton, rice,and canola.
 13. A population comprising at least 10 syntheticpreparations of claim 1 within a packaging material.
 14. A method ofaltering a trait in an agricultural plant seed or an agricultural plantgrown from said seed, said method comprising inoculating said seed witha composition comprising endophytes capable of promoting germination andan agriculturally-acceptable carrier, wherein the endophyte replicateswithin at least one plant tissue and colonizes the cortical cells ofsaid plant.
 15. The method of claim 14, wherein said endophyte colonizesat least 1% of the cortical cells of said agricultural plant.
 16. Themethod of claim 14, wherein the altered trait is an improved functionaltrait selected from the group consisting of increased yield, fasterseedling establishment, faster growth, increased drought tolerance,increased heat tolerance, increased cold tolerance, increased salttolerance, increased tolerance to Fusarium infection, increasedtolerance to Puccinia infection, increased biomass, increased rootlength, increased fresh weight of seedlings, increased plant vigor,nitrogen stress tolerance, enhanced Rhizobium activity, enhancednodulation frequency, and early flowering time.
 17. The method of claim14, wherein the altered trait is a seed trait selected from the groupconsisting a greater germination rate, faster dormancy breakdown,increased energy of germination, increased seed germination vigor orincreased seed vitality than a population of control agricultural seeds.18. The method of claim 17, wherein the altered trait is reaching 50%germination faster than a population of control agricultural seeds. 19.The method of claim 14, wherein the endophytes are a selected from thegroup consisting of a spore-forming endophyte, a facultative endophyte,a filamentous endophyte, an endophyte capable of living within anotherendophyte, an endophyte capable of forming hyphal coils within theplant, an endophyte capable of forming microvesicles within the plant,an endophyte capable of forming micro-arbuscules within the plant, anendophyte capable of forming hyphal knots within the plant, an endophytecapable of forming Hartig-like nets within the plant, and an endophytecapable of forming symbiosomes within the plant.
 20. The method of claim14, wherein the altered trait is altered gene expression, wherein thegene is selected from the group consisting of a gene involved in plantgrowth, an acquired resistance gene, and a gene involved in protectionfrom oxidative stress.